RU2630974C1 - Conjugate of antitumour drugs with recombinant alpha-fetoprotein and its functional fragments and method of their producing - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: it is obtained as particles of copolymers of lactic and glycolic acids with antitumour cure of anthracycline series included in them by interaction of -COOH groups of particles activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide with at least 5-fold excess relative to carboxyl groups, with amino groups of the recombinant alpha-fetoprotein having sequences selected from the group: SEQ1, SEQ2, SEQ3, SEQ4, not less than equimolar amounts in relation to activated carboxyl groups, at a pH from 7.4 to 9, followed by purification on a column of Superose 12 carrier with complete separation of reaction products and separation of the desired conjugate in pure form. A method of producing the conjugate is also disclosed.

EFFECT: increase the effectiveness of the antitumour drug.

3 cl, 7 ex, 10 dwg

Description

The invention relates to the field of medicine, biology, biotechnology, molecular biology, nanobiotechnology and pharmacology, and is a conjugate and methods for their preparation, consisting of cytostatics, biodegradable polymer, surfactant, cryoprotectant and vector molecules for targeted delivery of cytostatics to tumor cells.

Antitumor preparations of the anthracycline series are used as cytostatic preparations, selected from the group: (doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, aclarubicin, zorubicin, pyrarubicin, valrubicin), as a biodegradable polymer as a copolymer as an active glycol copolymer substances - polyvinyl alcohol of various concentrations, as a cryoprotectant - mono- and disaccharides, as vector molecules - recombinant protein molecules alpha-fetoprotein a (AFP) with and without His and Tag sequences and / or a fragment of a recombinant protein molecule of alpha-fetoprotein (AFP). The conjugates are in the form of lyophilisates intended for intravenous administration and provide directed and prolonged action.

The invention improves targeting for tumor cells, which increases the effectiveness of treatment and reduces overall toxicity.

A way to solve the above problems is to develop a system of targeted (vector) delivery of chemotherapeutic drugs in the form of polymer or liposomal forms associated (conjugated) with vector molecules having a specific affinity for the surface of tumor cells.

Famous work Farokhzad O.S. et al., which presents a system for targeted delivery of anticancer drugs based on polymer particles of PLGA-PEG-COOH to prostate cancer cells. The aptamer A10 PSMA, which selectively binds the extracellular region of the prostate-specific antigen, served as a vector molecule [1].

Known technical solutions protected by patents US 20130052134, WO 2013127949, CN 103745793, CA 2648099, which describe conjugation methods using various carbodiimide derivatives and where copolymers such as PEG-PLGA-PLL, DSPE-PEG-COOH, PLGA-b are used -PEG, etc.

The disadvantage of these methods is the relatively narrow scope.

Technical solutions are also known, protected by patents CN 101744789, PT 1539976, WO 2010028217, US 7105158, US 6555110, which describe methods of conjugation using carbodiimides.

The disadvantage of these inventions is the relatively large duration of the main reaction.

Also known is the method protected by US patent 7163698 B2, which is the synthesis of a PLGA polymer using peptide vector molecules and an activating agent EDC, in which organic phases are used during the conjugation reaction to obtain a modified polymer, after which polymer particles are obtained.

There are also known methods for the preparation of conjugates of antitumor drugs with antibodies acting as vector systems [patents WO 2016036794, WO 2014119624], in which antibodies are used as vector molecules.

The disadvantage of these inventions is the fact that the used vector molecules do not provide effective targeting against tumor target cells, and are also characterized by a relatively low concentration of the drug in the final conjugate. In addition, the methods are characterized by relatively high complexity, caused by the need to introduce an additional linker to obtain a vector system.

There are also works that present the results of the use of bombesin (BBN) as a vector (Hitesh et al., 2014), a system of targeted delivery based on polymer particles using monoclonal antibodies (Li et al., 2011) [2, 3] . In a number of other studies, human alpha-fetoprotein (AFP) protein is used as a vector molecule for the delivery of antitumor drugs, because it has receptors on the surface of tumor cells [4]. The RF patents provide data on the development of a number of AFP-based drugs and methods for their preparation from natural sources [RU 2121350 C1, 11/10/1998; RU 2123009 C1, 12/10/1998; RU 2154468 C1, 11/09/1999; RU 2105567 C1, 02.27.1998].

US20090068115 describes a method for activating carboxyl groups of polymer particles using EDC, for which a reaction is carried out in 0.1 M TEMED buffer, pH 4.8, as well as further purification using dialysis.

The disadvantage of this method is the relatively large time costs.

US2010015051 discloses a method for conjugation in aqueous media in borate buffer at slightly acidic pH values, the excess transferrin protein being removed by ultracentrifugation.

The disadvantage of this method is the relatively large complexity and relatively large time costs.

In the patent of the Russian Federation [RU 2317102 C1, A61K 38/08, 02/20/2008] the use of the QMTOVNOG peptide as an analogue of the alpha-fetoprotein fragment is described as a vector molecule. In particular, it is proposed the use of a peptide of the formula QMTOVNOG, which is an analogue of the fragment of α-fetoprotein from the 472th to 479th amino acids, which can be selectively captured by tumor cells, as a vector molecule for targeted delivery of antitumor drugs to tumor cells. In addition, a conjugate of a peptide of the formula QMTOVNOG with doxorubicin is proposed, in which doxorubicin is covalently attached to the indicated peptide via a thioether bond in which the covalent thioether bond between them is created by reacting the SH group of the derivative peptide obtained by the reaction of the peptide with N-hydroxysuccincimide amide acid, and a double bond of the maleimide group of hydrazone doxorubicin obtained by reaction of doxorubicin with 4- (N-maleimidomethyl) cyclohexane-1-carbonylhydrazide. Also proposed is a pharmaceutical composition for the treatment of cancer, comprising a doxorubicin conjugate with a vector molecule in an effective amount and a pharmaceutical carrier suitable for intravenous administration, characterized in that it contains the conjugate of doxorubicin conjugate.

The disadvantage of this conjugate is its relatively low targeting for target tumor cells.

One of the close ones to the proposed targeted delivery system is an antitumor peptide preparation based on the recombinant human alpha-fetoprotein (AFP) fragment. The drug is able to bind to the alpha-fetoprotein receptor and inhibit estradiol-induced cell growth of hormone-dependent tumors, and also perform the function of a vector molecule for targeted delivery of a chemotherapeutic drug to tumor cells [RU 2285537 C1, AK 38/12, 10.20.2006].

The disadvantage of this drug is the relatively low efficiency of use.

Closest to the proposed drug is an antitumor drug [RU 2451509 C1, A61K 31/337, 05/27/2012], which is a stable nanoparticle and includes a cytostatic agent, biodegradable polymer, surfactant, cryoprotectant and vector molecule for targeted delivery of particles to the affected organs and tissue, while as a cytotoxic agent contains paclitaxel - 0.4 ÷ 1.0 wt.%, as a biodegradable polymer - a copolymer of lactic and glycolic acids (PLGA 50:50) - 14.0 ÷ 15.0 wt.%, as a surfactant, su vinyl chloride polyvinyl alcohol - 3.5 ÷ 4.0 wt.%, as a vector molecule for targeted delivery of particles to the affected organs and tissues - the C-terminal domain of alpha-fetoprotein (r3dAFP) - 0.1 ÷ 0.3 wt.% and as a cryoprotectant - D-mannitol - 75.0 ÷ 80.0 wt.%.

The disadvantage of the closest technical solution regarding the proposed antitumor drug is the relatively low efficiency, due to the relatively low targeting against tumor cells, which reduces the effectiveness of the treatment and increases the overall toxicity in its application.

Closest to the proposed method is a method for producing a conjugate of a polymer-encapsulated antitumor agent from a group of taxanes with an active AFP fragment described in the same technical solution [RU 2451509 C1, A61K 31/337, 05.27.2012], in particular, a method for producing a conjugate of a milk and glycol copolymer acids with doxorubicin, according to which 150.0 mg of a copolymer of lactic and glycolic acids (PLGA-COOH 50/50) containing a free terminal carboxyl group is dissolved in 1.5 ml of chloroform, 18.0 mg of dicyclohexylcarb are added to the solution odiimide and 10.0 mg N-hydroxysuccinimide (10-fold molar excess with respect to the polymer), the reaction mixture is stirred for 2 hours at room temperature and separated from the precipitated dicyclohexylurea by filtration, 5.0 μl of triethylamine and 7.5 mg are added to the mixture doxorubicin hydrochloride, the reaction mixture is stirred for 15 hours, the reaction mixture is purified from excess doxorubicin by three times extraction with 0.1 M aqueous hydrochloric acid, the reaction product is purified by precipitation in ice-cold m ethanol, the precipitate is collected by centrifugation at 18 thousand g for 30 minutes, after having previously evaporated from a solution of chloroform under vacuum.

The disadvantage of the closest to the proposed method is the relatively narrow scope, due to the fact that it cannot be used to obtain an antitumor drug with higher efficacy, which is determined by increased targeting against tumor cells and lower toxicity when it is used.

In addition, the known method is characterized by a relatively long reaction time. Additionally, it can be noted that since the purification operation in the known method consists in centrifugation and ultrafiltration, the resulting conjugate can be separated only from low molecular weight impurities and it is impossible to separate unbound protein molecules, which can lead to a decrease in the effectiveness of the drug due to blocking of receptors on the surface of tumor cells, which in turn, leads to a decrease in the level of binding of the target drug.

The problem that is solved in the proposed invention is the development of an antitumor drug with higher efficacy due to increased targeting against tumor cells and reduced toxicity in its use, as well as its production method, which has a wider scope, shorter time to obtain a cleaner the drug.

The technical result of the claimed invention is to expand the arsenal of drugs with proven antitumor activity and methods for their preparation by obtaining an antitumor drug with higher efficacy due to increased targeting against tumor cells and lower toxicity when it is used, as well as its production method, which has a wider scope, shorter time to obtain a cleaner product.

The problem is solved, and the required technical result is achieved by the fact that the proposed conjugate with antitumor activity, upon receipt of which provide the interaction of particles from copolymers of lactic and glycolic acids with antitumor drugs included in them anthracycline series, with -COOH groups activated by 1-ethyl- 3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide with at least 5-fold excess of them in relation to carboxyl groups, with amino groups of recombinant protein a pf-fetoprotein having sequences selected from the group: SEQ1, SEQ2, SEQ3, SEQ4, in equimolar amounts with respect to carboxyl groups, at a pH of 7.4 to 9, and also carry out subsequent purification on a column with a Superose 12 carrier and carry out the separation of reaction products and isolate the target conjugate in pure form.

In addition, the required technical result is achieved by the fact that, as included in the copolymers of lactic and glycolic acid antitumor preparations of the anthracycline series, either doxorubicin, or daunorubicin, or ipirubicin, or idarubicin, or mitoxantrone, or aclarubicin, or zorubicin, or pira valrubicin.

In addition, the required technical result is achieved by the fact that to obtain the conjugate, a method is used, characterized in that the COOH groups of polymer particles activate 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide with at least 5-fold excess of in relation to carboxyl groups, add no more than 2 μl of 2-mercaptoethanol and incubate for 10 minutes, then add recombinant protein not less than in equimolar amounts with respect to activated carboxyl groups, adjust the pH of the reaction mixture to intramural values (from 7.4 to 9), left to mix for 30 minutes, then stop the reaction and add no more than 2 μl of ethanolamine, after which the reaction mixture is purified on a Superose 12 column with a complete separation of the reaction products and the target conjugate is isolated in pure form.

The drawing graphic materials presented:

in FIG. 1 - level of binding (+ 4 ° C) and endocytosis (+ 37 ° C) by tumor cells of the MCF7 human breast adenocarcinoma after 1 hour of incubation with proteins: AFP abortion and recombinant AFP;

in FIG. 2 is a chromatogram for the separation of conjugation reaction products of polymer particles containing an antitumor preparation of the anthracycline series and a protein vector molecule using size exclusion chromatography on Superose 12 resin;

in FIG. 3 - level of binding (+ 4 ° C) and endocytosis (+ 37 ° C) by tumor cells of the P388 mouse lymphocytic leukemia after 1 hour of incubation with a conjugate of nanoparticles containing doxorubicin, with a vector protein (AFP-Fitc) and unbound vector protein (AFP- Fitc);

in FIG. 4 - level of binding (+ 4 ° C) and endocytosis (+ 37 ° C) by HeLa human cervical carcinoma tumor cells after 1 hour of incubation with nanoparticles and conjugate of nanoparticles containing doxorubicin with vector protein (AFP3D);

in FIG. 5 - cell survival (IC50, doxorubicin concentration μM) of MCF-7 human breast adenocarcinoma after incubation with the drug substance doxorubicin (DR) and the conjugate of polymer particles of doxorubicin with rAFP (rAFP-nano-DR);

in FIG. 6 is a comparison of IC ₅₀ values (doxorubicin concentration, nM) for human peripheral blood lymphocyte cells after 72 hours of incubation with doxorubicin, nanoparticles and a conjugate of nanoparticles containing doxorubicin, with vector protein (rAFP);

in FIG. 7 - amino acid sequence of recombinant AFP (SEQ1);

in FIG. 8 - amino acid sequence of a recombinant fragment of AFP (AFP3D) (SEQ2);

in FIG. 9 - amino acid sequence of recombinant AFP with affinity tag his-tag (AFP-His) (SEQ3);

in FIG. 10 is the amino acid sequence of recombinant AFP affinity tag FLAG-tag (underlined) (AFP-Flag) (SEQ4).

A method of obtaining a conjugate of antitumor drugs with recombinant alpha-fetoprotein and its functional fragments is as follows.

Obtained by the proposed method, the antitumor drug provides targeted delivery of antitumor drugs directly to the tumor cells, protecting them from the action of resistant mechanisms and providing a prolonged effect.

The claimed method is intended to produce conjugates of polymer particles containing cytostatic preparations with recombinant AFP and its functional fragments.

To obtain the conjugates used carbodiimide method. This method is notable for its simplicity, relatively short reaction time, and also relatively acceptable conditions in which it proceeds.

Note that, one of the ways to treat cancer is the use of chemotherapeutic drugs. Over the years, anthracycline antibiotics have been used as antitumor substances [5]. The first anthracyclines were isolated from the pigment-producing bacteria Streptomyces peucetius in the 1960s. These drugs are actively used in the treatment of malignant tumors of various origins, such as various hematological types of oncology, soft tissue sarcoma, carcinoma and other tumors. The spectrum of indications for the use of a particular antibiotic is determined by its chemical structure, individual pharmacokinetic and pharmacodynamic properties and its degree of knowledge.

The mechanism of action of drugs of this group is due to the inhibition of synthesanucleic acids by intercalation between pairs of nitrogen bases, which leads to disruption of the secondary helixing of DNA due to interaction with topoisomerase II. In addition, there is an interaction with lipids of cell membranes, thereby disrupting ion transport and a number of other cell functions.

The present invention relates to therapeutic combinations containing anthracycline drugs. These include the following drugs, not limiting the scope of the invention:

1. “Doxorubicin” is an anthracycline antibiotic. It is used to treat lymphoblastic leukemia, soft tissue sarcoma, osteogenic sarcoma, Ewing sarcoma, breast cancer, thyroid cancer, Wilms tumor, neuroblastoma, bladder cancer, stomach cancer, ovarian cancer, lymphogranulomatosis, non-Hodgkin’s lymphoma, trophoblastic cancer ovary, Kaposi’s sarcoma [6-10].

The mechanism of action of doxorubicin is the interaction with DNA, the formation of free radicals and direct effects on cell membranes with inhibition of nucleic acid synthesis. Cells are sensitive to the drug in S and G2 phases. However, due to the high toxicity of doxorubicin, its use is associated with a number of side effects: thrombocytopenia, leukopenia, anemia, cardiomyopathy, heart failure, arrhythmia, stomatitis, esophagitis, nausea, vomiting, diarrhea, azoospermia, amenorrhea, nephropathy, allergy, allergy, and allergy and etc.

2. “Daunorubicin” is used for the treatment of acute lymphoblastic and non-lymphoblastic (myeloblastic, monoblastic, erythroblastic) leukemia, non-Hodgkin’s lymphomas, Ewing's tumor, Wilms’s tumor, blast crisis of chronic myelogenous leukemia, lymphosarcoma, adult uterine chorionoid cell carcinoma, soft tissues, neuroblastomas (in children). Used in combination with other antitumor agents as part of remission induction programs [11, 12].

The mechanism of action of daunorubicin is the effect on the S-phase of mitosis. In addition, daunorubicin blocks the matrix function of DNA, disrupting the synthesis of nucleic acids and protein. Antitumor activity is also realized through interaction with membranes and the formation of free radicals of the semiquinone type.

Daunorubicin has a number of side effects, which include the following: inhibition of bone marrow hematopoiesis (anemia, leukopenia, neutropenia, thrombocytopenia), the development or worsening of heart failure (arrhythmia, shortness of breath, swelling in the ankle joints), nausea, and decreased appetite. ulceration of the gastrointestinal mucosa (ulcerative stomatitis, esophagitis), hyperuricemia (painful or difficult urination, cystitis), amenorrhea, azoospermia, allergic reactions, headache, alopecia, etc.

Thus, a significant drawback of the antitumor drugs currently used is their high toxicity, which causes intoxication of the patient's body, as well as multiple side effects.

Another disadvantage is the occurrence of multidrug resistance (MDR) in tumor cells. The MDR mechanism is associated with the overexpression of transmembrane proteins of the ABC transporter family, which are capable of removing chemotherapeutic agents from tumor cells [13].

The invention can be applied for the treatment of tumor malignant neoplasms in the presence of alpha-fetoprotein (AFP) receptors on the surface of tumor cells, namely: for the treatment of human breast adenocarcinoma of the MCF 7 lines and subline MCF 7 Adr resistant to anthracycline antibiotics; human cervical carcinomas of the HeLa line; human osteosarcoma line MG 63; human ovarian adenocarcinomas of the SKOV3 line, resistant SKVLB line, and OVCAR 8 line; Human B-cell lymphomas of the lines Raji and Namalva; Human T-cell lymphomas of the lines CEM, Molt-4, Jukart, QOS; human erythroleukemia line K562; human myeloid leukemia of the TF-1 and KG-1 lines; human prostate adenocarcinomas of the LnCaP and Du145 lines; human epidermoid carcinoma A-431; human neuroblastoma line IMR-32; human hepatomas of the Alexander and HepG2 line; Colo320 human sigmoid colon carcinoma; Caco-2 human colon adenocarcinomas; HuTu80 human duodenal adenocarcinoma; rectal adenocarcinoma of the human line SW837; non-small cell lung carcinoma of the human line A549; human gliomas of the line U373MG; human myelomas of the lines U266 and IM9; human leukosarcoma line SK-UT-1B; human pharyngeal carcinomas of the KB line; small cell lung carcinoma of the human line H69; human melanomas of the lines SK-MEL-1, MS and Bro B19.

The terms "cancer" and "malignant tumor" refer to or describe the physiological condition in mammals, which is usually characterized by unregulated cell growth / proliferation.

Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such malignancies include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, cancer of the rectum and colon, endometrial or uterine carcinoma, salivary carcinoma, kidney cancer, liver cancer, cancer is simple you, vulvar cancer, thyroid cancer, liver carcinoma and various types of head and neck cancer.

The term "antitumor activity" means the activity of the conjugate in relation to both hormone-dependent and hormone-independent tumors.

The terms "chemotherapeutic drugs", "antitumor drugs", "cytotoxic agent" denote chemical compounds suitable for the treatment of cancer, with a similar mechanism of action, structural formula and physico-chemical properties. These terms include antitumor antibiotics of the anthracycline series or other intercalating agents that meet the above requirements (such as: doxorubicin, daunorubicin, etc., not including dactinomycin).

A “chemotherapeutic agent” is a chemical compound that can be used to treat a malignant tumor. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carboxwon, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethyl melamine; acetogenins (in particular, bullatacin and bullatacinon); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogue of topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetyl camptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callistatin; CC-1065 (including its synthetic analogs, adozelesin, carzelesin and biselezin); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pankratistatin; sarcodiktiin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, holophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembikhine, phenesterol, prednimustine, trophosphamide, uramustine; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediin antibiotics (e.g. calicheamicin, in particular calicheamicin gamma II and calicheamicin omega II (see, for example, Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynamin, including dinemycin A ; esperamycin; as well as the neocarcinostatin chromophore and related chromophores of chromoproteins - enediin antibiotics), aclacinomisins, actinomycin, autramycin, azaserin, bleomycin, cactinomycin, carabicin, carminomycin, ocrominomycin-5, -dominocinomycin, L-norleucine, doxorubicin ADRIAMICIN® (including I am morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin C, mitomycin cytomycinomycidiricinomycinomycidiricinomycinomycinomycinphybrinomycinomycinomycinphybrinomycinomycinomycinphybrinomycinomycinomycinomycinomycin ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimerexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, karmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, phloxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostan, testolactone; adrenal suppressants, such as aminoglutethimide, mitotane, trilostane; a folic acid compensator such as folinic acid; Aceglaton; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisanthrene; edatraxate; defofamine; demecolcin; diazikhon; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pyrarubicin; losoxantrone; 2-ethyl hydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spiro germanium; tenuazonic acid; triaziquon; 2,2 ', 2-trichlorotriethylamine; trichotecenes (in particular, T-2 toxin, verracurin A, roridin A and anguidin); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacitosin; arabinoside ("ara-C"); thiotepa; taxoids, for example, paclitaxel TAXOL® (Bristol-Myers Squibb Oncology, Princeton, NJ), a cremophor-free paclitaxel preparation based on engineered albumin-linked nanoparticles ABRAXANE ™ (American Pharmaceutical Partners, Schaumberg, Illinois) and doxetaxel TAXOTERElen® (Rhone Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovorin; vinorelbine (NAVELBINE®); novantron; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids and derivatives of any agent specified above; as well as combinations of two or more of the above, such as CHOP, an abbreviation for combination therapy with cyclophosphamide, doxorubicin, vincristine and prednisone, and FOLFOX, an abbreviation for oxaliplatin treatment regimen (ELOXATIN ™) in combination with 5-FU and leucovorin.

Also included in this definition are anti-hormonal drugs that act by regulating, decreasing, blocking or inhibiting the effects of hormones that can stimulate the growth of a malignant tumor, and are often used in the form of systemic treatment or treatment at the level of the whole organism. They themselves can be hormones. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen NOLVADEX®), raloxifene EVISTA®, droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifen, LY117018, onapriston and TOREMEN; antiprogesterones; estrogen receptor downregulators (ERD); agents that function to suppress or stop ovarian function, for example, luteinizing hormone releasing hormone agonists (LHRH), such as LUPRON® and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetate and trypterelin; other antiandrogens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the aromatase enzyme, which regulates the production of estrogen in the adrenal glands, such as, for example, 4 (5) -imidazoles, aminoglutetimide, megestrol acetate MEGASE®, exemestane AROMASIN®, formestane, fadrozole, Vorozol RIVISOR®, letrozole FEM and anastrozole ARIMIDEX®. In addition, this definition of chemotherapeutic agents includes bisphosphonates such as clodronate (e.g., BONEFOS® or OSTAC®), ethidronate DIDROCAL®, NE-58095, zoledronic acid / zoledronate ZOMETA®, alendronate FOSAMAX®, pamidronate AREDIA SKELID® or tiloud risedronate ACTONEL®; as well as troxacitabine (a 1,3-dioxolane nucleoside analog of cytosine); antisense oligonucleotides, in particular oligonucleotides, which inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example ALLOVECTIN® vaccine, LEUVECTLN® vaccine and VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitor; rmRH ABARELIX®; lapatinib ditosylate (low molecular weight double tyrosine kinase inhibitor ErbB-2 and EGFR, also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any of the above.

Examples of the implementation of the proposed invention.

Example 1. Obtaining biomass of a recombinant AFP fragment (r3D AFP).

Work with bacterial strains was carried out under a laminar under sterile conditions. For transformation, competent Bl21 (DE3) cells from Stratogen were used. The cell tube (0.1 ml) was thawed on ice, 5 μl of plasmid solution was added at a concentration of 20 ng / ml and incubated for 10-15 minutes on ice, after which it was subjected to heat shock at 42 ° C for 2 minutes. Then the tube was placed on ice for 30 s, then 1 ml of LB medium was added and incubated for 30-45 minutes at 37 ° C with stirring. The cells were then pelleted by centrifugation (2000 g, 5 minutes), 1 ml of supernatant was removed, the pellet was resuspended in the remaining medium and plated on Petri dishes with agarized LB medium containing ampicillin (100 μg / ml). 2-3 transformant colonies were placed in 10 ml of LB medium containing ampicillin (100 μg / ml) and incubated at 37 ° C with vigorous stirring overnight. From the obtained liquid culture, plating was performed on a Petri dish with agarized LB medium containing ampicillin (100 μg / ml). The cup was incubated at 37 ° C overnight. Of the grown colonies, 6 of the largest were selected, each of which was placed in 10 ml of LB medium with the addition of ampicillin and incubated at 37 ° C. Upon reaching an optical density of OD ₆₀₀ from 0.5 to 1.2 units, IPTG (isopropyl-β-D-1-thiogalactopyranoside) was added to a final concentration of 0.1 to 2 mM and incubation was continued for 3 hours, after which the expression level of the target protein was determined by electrophoresis in page. The resulting biomass is centrifuged for 15 minutes at 10,000 RPM.

Example 2. Purification and isolation of a recombinant AFP fragment (r3D AFP).

Wet biomass (6 g) was resuspended in 100 ml pH = 8.0 buffer containing 50 mM sodium phosphate and 2 tablets of protease inhibitors without EDTA (Roche). Then lysozyme was added to a concentration of 50 mg / L and incubated for 10 min at room temperature, stirring with a glass rod. Next, ultrasonic treatment was performed 7 times for 40 strokes at 0 ° C. Sodium deoxycholate was then added to 0.2 wt.%, And stirred for 10 minutes at room temperature. Then it was centrifuged for 10 min at 10,000 rpm. The soluble fraction was collected. The precipitate was washed several times with phosphate buffer and centrifuged at 10,000 rpm. The sediment of inclusion bodies and the soluble fraction were stored at -20 ° C. The sediment of inclusion bodies was resuspended in 10 ml of solubilizing buffer (150 mM NaCl, 10 mM KH ₂ PO ₄ , 8 M urea, 12 mM mercaptoethanol, pH 7.4) and left under stirring at room temperature for 1 hour. The resulting solution was centrifuged (10,000 rpm, 10 min) and the supernatant was further used for refolding. Five volumes of a renaturing buffer (150 mM NaCl, 10 mM KH ₂ PO ₄ , 1 mM GSSG, 5 mM GSH, pH 8.5, + 4 ° C) were preliminarily passed through a Superose 12-supported column. Then dilutions of the solubilizing buffer were prepared by renaturing, 0.5 ml each, to the final concentration of urea in a solution of 8, 6, 4, 2, and 0 M and mercaptoethanol 12, 9, 6, 3, 0 mM, respectively, and applied to the column, starting with a solution with the lowest concentration of denaturing agents. On the prepared column, 2 ml of solubilized r3D AFP (k) (not more than 20 mg) was applied, then 0.5 ml of solubilizing buffer, and the target protein was eluted with 3 volumes of renaturing buffer. The obtained fraction was dialyzed against a 1000-fold volume of phosphate-buffered saline at pH = 8.5 and stored at -70 ° C. The recombinant protein was further purified by metal chelate chromatography on a carrier containing Ni ^{2+ ions} and, if necessary, concentrated using concentration cells.

Example 3. Obtaining biomass of recombinant AFP (AFP-His, AFP-Flag, AFP).

Work with yeast strains was carried out under a laminar under sterile conditions. We used yeast strains Pichia Pastoris GS115 and KM 71 transformed with genetic constructs based on commercial plasmids pPIC9K and pPICZ A carrying the nucleotide sequence encoding human full-length AFP, as well as human AFP with additional amino acid inserts (his-tag and flag-tag). Transformants were plated on plates with agarized YPD medium containing geniticin (in the case of AFP) or zeocin (in the case of AFP-His, AFP-Flag) at a concentration of 50 μg / ml. After three days of incubation at 28 ° C, recombinant clones were transferred to fresh antibiotic plates, grown and plated in tubes with 5 ml of BMGY medium. The tubes were incubated on a rotary shaker for 16 hours at 28 ° C, after which the biomass was precipitated by centrifugation. Cells were washed with saline, then resuspended in 5 ml of BMMY medium. The induced cells were incubated on a rotary shaker for 6 days, while methanol was added daily to a concentration of 1%. The resulting biomass was centrifuged at 3000 RPM for 15 min, after which the supernatant was decanted from the sediment. 0.1% azide was added to the resulting culture fluid and stored at -70 ° C.

Example 4. Purification and isolation of recombinant AFP (AFP-His).

AFP-His culture fluid, after building up in a 1 L shaker, is centrifuged for 10 min at 3000 rpm + 4C. The supernatant is decanted, the pH is adjusted to 7.4 with 10 M NaOH, after which salts are precipitated, which are disposed of by centrifugation for 10 min at 7000 rpm, then the supernatant is gently decanted. Imidazole is added to the resulting supernatant to a final concentration of 25 mM. The Ni-Sepharose column was equilibrated with 1 * PBS, after which the resin was transferred to a flask with QOL and stood for 1 hour at room temperature on a rocking chair with slight stirring. After 1 h of stirring, the QOL with the resin was transferred to 50 ml plastic Falcons, after which the resin was precipitated by centrifugation for 2 min at 500 rpm at room temperature. After centrifugation, the entire resin is transferred to a column. After adding the resin to the column, it is washed with a solution (1 * PBS containing 25 mM imidazole and 0.5 M NaCl), then elution with a solution (1 * PBS containing 0.5 M imidazole and 0.5 M NaCl) is carried out. The purified protein must be dialyzed during the day, after which the protein is lyophilized with 1% D-mannitol in the total protein volume during the day. The resulting lyophilisate was concentrated 10 times, 0.1% azide was added and stored at -70 ° C.

Example 5. Purification and isolation of recombinant AFP (AFP-Flag, AFP).

The culture fluid in a volume of 1 l is passed through a column with a carrier of Br-CN-sepharose conjugated with antibodies to AFP, during the day at + 4 ° C. Then, at room temperature, it is washed with 10 volumes of 1 × PBS, after which 3M NaSCN is eluted. The purified protein must be dialyzed during the day, after which the protein is lyophilized with 1% D-mannitol in the total protein volume. The resulting lyophilisate was concentrated 10 times, 0.1% azide was added and stored at -70 ° C.

Example 6. Obtaining a conjugate of polymer particles containing a cytostatic preparation with recombinant proteins (AFP-His, AFP-Flag, AFP, r3D AFP).

To 25 mg of polymer particles obtained in a known manner using PLGA-COOH (14, 15, 16), previously washed from excess surfactant dissolved in 1 * PBS, add at least a 5-fold excess of EDC and NHS with respect to the number of carboxyl groups (concentration of solutions 10 mg / ml in 1 * PBS) and stirred for 15 minutes After that, no more than 2 μl of 2-mercaptoethanol is added to the reaction mixture and incubated for 10 minutes, then the recombinant protein is added to the activated polymer particles at least in equimolar amounts with respect to the activated carboxyl groups and the pH of the reaction mixture is adjusted to alkaline values (from 7.4 to 9), then left to mix for another 30 minutes. To stop the reaction, no more than 2 μl of ethanolamine (or any amino derivative with similar properties) is introduced, after which the reaction mixture is purified on a Superose 12 column with a complete separation of the reaction products, and thereby, isolation of the target conjugate in pure form. The purified conjugate is lyophilized with the addition of a cryoprotectant during the day, after which it is stored as a lyophilizate at -70 ° C.

Example 7. Determination of levels of cytotoxic activity.

Tumor cells were cultured in DMEM medium, in plastic culture bottles containing 10% fetal bovine serum and 50 μg / ml gentamicin, in a CO ₂ incubator at 37 ° C in a humidified atmosphere containing 5% CO ₂ . Cells were scattered 2 times a week using Versen's solution. Mononuclear leukocytes of peripheral blood of healthy volunteers were isolated by centrifugation of blood through a gradient of ficoll-pack solution according to the method

To assess cytotoxic activity, tumor cells were seeded in 96-well plates of 5 thousand cells per well one day before the experiment. Mononuclear leukocytes of peripheral blood of healthy volunteers were isolated by centrifugation of blood through a gradient of ficoll-pack solution according to the Boyum method and scattered into 96-well plates at 270 thousand cells per well on the day of the experiment. The preparations were added to the cells in triplets and incubated under standard conditions. In the case of lymphocytes, they were cultured for 1 h, then PBS with pH 7.4 was washed 2 times, placed in 200 μl of DMEM with 10% FBS (lymphocytes were placed in RPMI1640 with 10% FBS) and incubated for 72 hours. Cell survival was determined using MTT test. 4 hours before the end of incubation, 50 μl of MTT solution at a concentration of 1 mg / ml in cell culture medium was added to each well. After color development, the medium was removed, the precipitated formazan crystals were dissolved in 100 μl DMSO, and the color intensity was measured by absorption at 540 nm using a plate photometer. Cell survival was evaluated as a percentage of the untreated control. OriginPro (OriginLab Corporation) was used to plot survival charts, calculate IC50 values, and statistically process the results.

Thus, in the proposed technical solution, the required technical result is achieved, which consists in expanding the arsenal of agents with proven antitumor activity and methods for their preparation, since the proposed method provides an antitumor drug with higher efficacy due to increased targeting against tumor cells and reducing toxicity when using it. The proposed drug provides targeted delivery of antitumor drugs directly to tumor cells, protecting them from the effects of resistant mechanisms and providing a prolonged effect.

BIBLIOGRAPHY

1. Farokhzad O.S. Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo / Farokhzad O.C., Cheng J., Teply B.A., Sherifi I., Jon S., Kantoff P.W. // Proceedings of the National Academy of Sciences - 103 (16) - 2006 - P. 6315-6320.

2. Hitesh Kulhari. Peptide conjugated polymeric nanoparticles as a carrier for targeted delivery of docetaxel / Hitesh Kulhari, Deep Pooja, Shweta Shrivastava et al. // Colloids and Surfaces B: Biointerfaces 117 - 2014 - P. 166-173.

3. Li Wang. Monoclonal antibody targeting MUC1 and increasing sensitivity to docetaxel as a novel strategy in treating human epithelial ovarian cancer / Li Wang, Hongmin Chen, Feng Hua Liu et al. // Cancer Letters - 2011 - Volume 300 - Issue 2, 28 - P. 122-133.

4. Moro R., Tcherkassova J., Song E. et al. // IVD Technology Magazine - 2005. - Vol. 11 - No. 5.

5. Weiss R. The anthracyclines: will we ever find a better doxorubicin / R. Weiss // Semin. Oncol. - 1992. - Vol. 19. - P. 670-686.

6. Gruber B. The effect of new anthracycline derivatives on the induction of apoptotic processes in human neoplastic cells. / B. Gruber, E. Anuszewska, W. Priebe // Folia Histochem. Cytobiol. - 2004. - Vol. 42. - P. 127-130.

7. O'Shaughnessy J. Liposomal anthracyclines for breast cancer: overview / J. O'Shaughnessy // Oncologist. - 2003. - Vol. 8. - P. 1-2.

8. Petrioli R. The role of doxorubicin and epirubicin in the treatment of patients with metastatic hormonerefractory prostate cancer / R. Petrioli, A. Fiaschi, E. Francini, A. Pascucci, G. Francini // Cancer Treat. Rev. - 2008. - Vol. 34. - P. 710-718.

9. Gewirtz D. A critical evaluation of the mechanism of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin / D. Gewirtz // Biochem. Pharmacol - 1999. - Vol. 57. - P. 727-741.

10. Breslow N. Doxorubicin for Favorable Histology, Stage II-III Wilms Tumor Results from the National Wilms Tumor Studies / N. Breslow, S. Ou, M. Beckwith, G. Haase, J. Kalapurakal, M. Ritchey, R. Shamberger, P. Thomas, G. D'Angio, D. Green // Cancer. - 2004. - Vol. 101. - P. 1072-1080.

11. Arcamone F. Doxorubicin. - London: Academic Press - 1981.

12. Daunorubicin hydrochloride Daunorubicin hydrochloride // European Pharmacopoeia. Fifth Edition: Monograph. - 2005 .-- S. 1389-1390.

13. Severin E.C. Problems and prospects of modern antitumor therapy / Severin E.S., Rodina A.V. // Successes in biological chemistry. - 200. - T. 46. - S. 43-64.

14. Betancourt T., Brown B., Brannon-Peppas L. Doxorubicin-loaded PLGA nanoparticles by nanoprecipitation: preparation, characterization and in vitro evaluation. Nanomedicine (Lond). 2007.2 (2): 219-32. (http // www.ncbi.nlm.nih.gov / pubmed / 17716122).

15. Chittasupho C., Lirdprapamongkol K., Kewsuwan P., Sarisuta N. Targeted delivery of doxorubicin to A549 lung cancer cells by CXCR4 antagonist conjugated PLGA nanoparticles. Eur J Pharm Biopharm. 2014.88 (2): 529-38 (http://www.ncbi.nlm.nih.gov/pubmed/25119723).

16. Park J, Fong PM, Lu J et al. PEGylated PLGA nanoparticles for the improved delivery of doxorubicin. Nanomedicine: nanotechnology, biology, and medicine. 2009.5 (4): 410-418. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2789916/).

MGYQELLEKCFQTENPLECQDKGEEELQKYIQESQALAKRSCGLFQKLGEYYLQNAFLVAYTKKAPQLTSSELMAITRKMAATAATCCQLSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGVGQCCTSSYANRRPCFSSLVVDETYVPPAFSDDKFIFHKDLCQAQGVALQTMKQEFLINLVKQKPQITEEQLEAVIADFSGLLEKCCQGQEQEVCFAEEGQKLISKTRAALGVLEHHHHHH

EAEARTLHRNEYGIASILDSYQCTAEISLADLATIFFAQFVQEATYKEVSKMVKDALTAIEKPTGDEQSSGCLENQLPAFLEELCHEKEILEKYGHSDCCSQSEEGRHNCFLAHKKPTPASIPLFQVPEPVTSCEAYEEDRETFMNKFIYEIARRHPFLYAPTILLWAARYDKIIPSCCKAENAVECFQTKAATVTKELRESSLLNQHACAVMKNFGTRTFQAITVTKLSQKFTKVNFTEIQKLVLDVAHVHEHCCRGDVLDCLQDGEKIMSYICSQQDTLSNKITECCKLTTLERGQCIIHAENDEKPEGLSPNLNRFLGDRDFNQFSSGEKNIFLASFVHEYSRRHPQLAVSVILRVAKGYQELLEKCFQTENPLECQDKGEEELQKYIQESQALAKRSCGLFQKLGEYYLQNAFLVAYTKKAPQLTSSELMAITRKMAATAATCCQLSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGVGQCCTSSYANRRPCFSSLVVDETYVPPAFSDDKFIFHKDLCQAQGVALQTMKQEFLINLVKQKPQITEEQLEAVIADFSGLLEKCCQGQEQEVCFAEEGQKLISKTRAALGVHHHHHH

EAEADYKDDDDKRTLHRNEYGIASILDSYQCTAEISLADLATIFFAQFVQEATYKEVSKMVKDALTAIEKPTGDEQSSGCLENQLPAFLEELCHEKEILEKYGHSDCCSQSEEGRHNCFLAHKKPTPASIPLFQVPEPVTSCEAYEEDRETFMNKFIYEIARRHPFLYAPTILLWAARYDKIIPSCCKAENAVECFQTKAATVTKELRESSLLNQHACAVMKNFGTRTFQAITVTKLSQKFTKVNFTEIQKLVLDVAHVHEHCCRGDVLDCLQDGEKIMSYICSQQDTLSNKITECCKLTTLERGQCIIHAENDEKPEGLSPNLNRFLGDRDFNQFSSGEKNIFLASFVHEYSRRHPQLAVSVILRVAKGYQELLEKCFQTENPLECQDKGEEELQKYIQESQALAKRSCGLFQKLGEYYLQNAFLVAYTKKAPQLTSSELMAITRKMAATAATCCQLSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGVGQCCTSSYANRRPCFSSLVVDETYVPPAFSDDKFIFHKDLCQAQGVALQTMKQEFLINLVKQKPQITEEQLEAVIADFSGLLEKCCQGQEQEVCFAEEGQKLISKTRAALGV

Claims (3)

1. A conjugate with antitumor activity based on a recombinant alpha-fetoprotein or its functional fragment and antitumor agent, characterized in that it is obtained in the form of particles from copolymers of lactic and glycolic acids with an antitumor agent included in them by the interaction of -COOH groups of particles activated 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide with at least 5-fold excess of them in relation to carboxyl groups, with amino groups of recombin an alpha-fetoprotein protein having sequences selected from the group: SEQ1, SEQ2, SEQ3, SEQ4, not less than in equimolar amounts with respect to activated carboxyl groups, at a pH of 7.4 to 9, followed by purification on a Superose 12 column with a complete separation of the reaction products and the isolation of the target conjugate in pure form. 2. The conjugate according to claim 1, characterized in that as an antitumor agent of the anthracycline series included in the copolymers of lactic and glycolic acids, it contains doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, aclarubicin, zorubicin, pirarubicin, val. 3. The method for producing a conjugate with antitumor activity according to claim 1, characterized in that the -COOH groups of polymer particles from copolymers of lactic and glycolic acids with the antitumor agent anthracycline included in them activate 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and When N-hydroxysuccinimide is at least 5-fold in excess with respect to the carboxyl groups, no more than 2 μl of 2-mercaptoethanol is added and incubated for 10 minutes, then a recombinant alpha-fetoprotein protein having the sequences selected from the group: SEQ1, SEQ2, SEQ3, SEQ4, not less than in equimolar amounts with respect to the activated carboxyl groups, adjust the pH of the reaction mixture to alkaline values from 7.4 to 9, leave it to mix for 30 minutes, then stop the reaction by not more than 2 μl of ethanolamine, after which the reaction mixture was purified on a Superose 12 support column with complete separation of the reaction products and the target conjugate was isolated in pure form.

Images