RU2435783C1 - Chimeric peptide and pharmaceutical composition for treating cancer - Google Patents

Abstract

FIELD: medicine, pharmaceutics. ^ SUBSTANCE: present invention refers to biotechnology and medicine. What is described is application of a chimeric peptide containing a functional 20-amino acid sequence from the p16INK4a cycline kinase inhibitor and a transport 16-amino acid sequence from the Antp protein for treating cancer selected from a group including colorectal cancer, renal cell carcinoma, lung cancer, breast cancer, bladder cancer, pancreatic cancer, uterine cancer, prostate cancer, stomach cancer and ovarian carcinoma with the transport amino acid sequence attached to an C-end of the specified functional sequence by means of groups X where X represents an amino acid sequence containing 1 - 50 amino acid residues. ^ EFFECT: invention can be used for creation of the preparation effectively penetrating into target cells and exibiting high cytostatic and cytotoxic action. ^ 4 cl, 19 dwg, 3 tbl

Description

FIELD OF THE INVENTION

The present invention relates to the field of biotechnology and medicine, in particular to a new chimeric peptide consisting of a cytotoxic p16 fragment with amino acid sequence 84-103 or 84-106 and an Antp transport fragment with amino acid sequence 43-58 having inhibitory activity against cyclin kinases. The invention also relates to the use of the aforementioned chimeric peptide for the treatment of cancer, as well as to a pharmaceutical composition having antiproliferative activity, and a method for treating cancer, comprising administering said chimeric peptide to a mammal in need of such treatment. The objective of the invention is to develop a drug that effectively penetrates into target cells and has a high cytostatic and cytotoxic effect.

State of the art

Since the beginning of the 90s in Russia more than 400 thousand cases of malignant neoplasms have been diagnosed annually in Russia (Davydov MI, Axel EM 2008). Moreover, in Europe, the annual mortality from cancer over the period from 1985 to 2002 continues to grow. In the structure of causes of mortality, oncological diseases take the 2nd place after diseases of the cardiovascular system. Therefore, the search for new medicinal antitumor drugs is one of the urgent tasks of modern biology and medicine.

Currently, a large number of works are devoted to the creation of new medicinal antitumor drugs based on the achievements of molecular biology (Richard JP et al., 2003; Takeshima K. et al., 2003; Jyotika A. et al., 2005, Kopnin B.P., 2000). It is generally accepted that a fundamental feature of a neoplastic cell is dysregulation of the cell cycle and apoptosis (Chappuis P. O., Kapusta L., 2005). It is known that the regulation of cell proliferation is controlled by sequential activation of cyclins and corresponding cyclin-dependent kinases (CDKs). The activity of cyclin kinases is determined by the expression level of the corresponding cyclins and the activity of specific cyclin kinase inhibitors (Kastan M.B., Bartek J., 2004). There are several families of cyclin kinase inhibitors. The most studied and practically important of them are p16INK4a, p21CIP / KIP, p27 KIP1 (Lowe S.W. et al., 2004). Mutations or hypermethylation of cyclin kinase inhibitor gene promoters are observed in 40-60% of cases of malignant lymphomas, pancreatic cancer and a number of other malignant neoplasms (Sawyers S., 2004; Ortega S. et al., 2002).

Based on these results, a number of low molecular weight cyclin kinase inhibitors were synthesized, some of which are being studied experimentally (Ross M.F., Murphy M.P., 2004), and one UCN-1 is in the first phase of clinical trials. Another possible direction for creating cyclin kinase inhibitors may be the use of functional sequences from the corresponding intracellular inhibitors (Ziegler A. et al., 2005). The p16INK4a protein is one of the most interesting candidates for the Cdk inhibitor group (Xu D. et al., 2004; Zhang Y. et al., 2005). The p16INK4a protein is known to inhibit cyclin-dependent kinases D and thereby the passage of the G1 phase of the cell cycle (Fu G.H. et al., 2005; Ben-Saadon R. et al., 2004). It has been shown that its function is impaired in a wide range of oncological diseases (Li J.Q. et al., 2004). Recently, experimental works have begun to appear that describe the use of the p16INK4a gene for gene therapy of tumors of various genesis (Lee AWC, Li JH et. Al. 2003; Liu SX, Tang SQ, Liang CY, 2003; Zhang Y., Liu J. et al . 2005). An additional motive for the search for technologies for the use of natural protein proliferation inhibitors was the discovery of short amino acid sequences (n = 15-30) that can perform vector (transport) functions in relation to peptide sequences and compounds of a different chemical nature (RNA, DNA) (Fawell S., Seery J. et al., 1994; Vives E., Brodin P., Lebleu B. 1997; Kaplan IM et al., 2005; Gupta B. et al., 2005; Fernandez-Carneado J. et al., 2005).

So far, the problem of restoring the impaired function of intracellular proteins has been tried to solve on the basis of gene delivery methods (gene therapy) (Georgiev G.P. 2000; Wender P.A. et al., 2000; Baryshnikov A.Yu. 2004). However, this technology has not yet gained widespread entry into clinical practice due to a number of fundamental problems.

An alternative way to solve this problem, based on the technology of peptide vectors with the ability to penetrate into cells without damaging the plasma membrane, is very promising due to the weak immunogenicity of such compounds and the ability to transfer large enough molecules.

The combination of the possibility of targeted delivery of peptides into the cell and the discovery of short functional domains in protein-regulators of various cellular functions created the prerequisites for the construction of molecules with a pathogenetic orientation (Schutze-Redelmeier M.R. et al., 2004; Trehin R., Merkle HP, 2004 ; Cong-Mei Wu et al., 2004). The relative simplicity of the synthesis of such molecules allows us to speak of the fundamental possibility of creating individual chemotherapy drugs based on them, i.e. affecting the pathological changes characteristic of this particular tumor (Perea S.E. et al., 2004).

The discovery of peptides capable of penetrating into the cell without the participation of membrane proteins and capable of carrying out intracellular transport of protein fragments and oligonucleotides associated with them, opens a new stage in the development of biology and medicine. One of the effective carriers of large molecules inside cells is the pAntp peptide. Its properties are known, in particular, from the publications of Derossi D. et al. The third helix of the Antennapedia homeodamain translocates through membranes.// J.Biol. Chem. 269 (1994) 10444-10450 and Morris MC. et al. A peptides carrier for the delivery of biologically active proteins in mammalian cells.//Nat. Biotechnology 19 (2001) 1173-1176.

According to the author, the closest analogue of the present invention is the patent US 6569833 B1 (Cyclacel Limited, GB). This document discloses peptides that bind to cyclin kinases and include amino acid residues 84-103 of the full-chain p16 protein and can be combined with the penetratin transport protein sequence via a disulfide bond formed between cysteine residues specifically attached to the C-terminus of p16 and N- end of the Antp peptide. The disadvantage of this approach is the need for selective and multi-stage synthesis of a chimeric molecule, which complicates the scheme for obtaining the target product and increases the overall time spent on synthesis.

Disclosure of invention

The present invention relates to the field of biotechnology and medicine.

The objective of the invention is the creation of a new chimeric peptide that has an increased therapeutic effect and does not require a complex and time-consuming scheme for its preparation.

The technical result of the present invention is an improved biomedical effect, objectively manifested in the pronounced cytotoxic effect of the indicated chimeric peptide in comparison with other similar peptides on tumor cells of diseases such as colorectal cancer, kidney cancer, lung cancer, breast cancer, bladder cancer, pancreatic cancer, uterine cancer, prostate cancer, stomach cancer and ovarian cancer, as well as simplifying the scheme for producing a chimeric peptide by reducing the total number steps Twa. The second technical result is the expansion of the arsenal of drugs for the treatment of cancer. In addition, the author of the present invention unexpectedly found a pronounced synergistic effect when using the above chimeric peptide with existing chemotherapeutic antitumor drugs (taxol and 5-fluorouracil).

The technical result is achieved by obtaining a chimeric peptide having a strictly defined sequence of binding of the functional and transport fragments in the chimeric peptide molecule, as well as by the presence of additional amino acid residues (from 1 to 50) that bind the two above-mentioned peptide fragments.

In one embodiment, the present invention relates to a chimeric peptide for the treatment of cancer, which contains a functional 20-amino acid sequence from a protein cyclin kinase inhibitor p16INK4a - DAAREGFLDTLVVLHRAGAR (SEQ ID NO.1), and a 16-amino acid transport sequence from Antp protein - RQIKIWFQNRRMWK (SEQ ID NO.2), wherein the amino acid transport sequence is attached to the C-terminus of the indicated functional sequence through group X, where X is an amino acid sequence nost comprising 1 to 50 amino acid residues.

In one preferred embodiment, the invention relates to a chimeric peptide that can be used to treat cancer selected from the group consisting of colorectal cancer, kidney cancer, lung cancer, breast cancer, bladder cancer, pancreatic cancer, uterine cancer, prostate cancer, stomach cancer, and ovarian cancer.

In a most preferred embodiment, the chimeric peptide can be used to treat colorectal cancer.

In another preferred embodiment, the chimeric peptide has the amino acid sequence DAAREGFLDTLVVLHRAGARSRQIKIWFQNRRMKWKK (SEQ ID NO.3).

In another preferred embodiment, the chimeric peptide has the amino acid sequence RGSDAAREGFLDTLVVLHRAGARSRQIKIW-FQNRRMKWKKSERKRGRQTYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLTE (SEQ ID NO.4).

In another embodiment, the present invention relates to the use of said chimeric peptide for the treatment of cancer.

In a preferred embodiment, the chimeric peptide is used to treat cancer selected from the group consisting of colorectal cancer, kidney cancer, lung cancer, breast cancer, bladder cancer, pancreatic cancer, uterine cancer, prostate cancer, stomach cancer and ovarian cancer .

In another embodiment, the present invention relates to the use of said chimeric peptide for the manufacture of a medicament for the treatment of cancer.

In a preferred embodiment, said medicament can be used to treat cancer selected from the group consisting of colorectal cancer, kidney cancer, lung cancer, breast cancer, bladder cancer, pancreatic cancer, uterine cancer, prostate cancer, and gastric cancer ovarian cancer.

In another embodiment, the present invention relates to a pharmaceutical composition having antiproliferative activity, which comprises, as a therapeutically active substance, the above chimeric peptide in combination with pharmaceutically acceptable carriers.

In another embodiment, the present invention relates to a pharmaceutical composition having antiproliferative activity, which contains two active substances, where the first active substance is the aforementioned chimeric peptide, and the second active substance can be a chemotherapeutic antitumor agent selected from the group consisting of taxol and 5 fluorouracil.

In another embodiment, the present invention relates to a method for treating an oncological disease, which comprises administering to the mammal in need of such treatment the above chimeric peptide.

In a preferred embodiment of the above method, the cancer is selected from the group consisting of colorectal cancer, kidney cancer, lung cancer, breast cancer, bladder cancer, pancreatic cancer, uterine cancer, prostate cancer, stomach cancer and ovarian cancer.

Brief Description of the Drawings

Figures 1 and 2 show the effect of pAntp chimeric peptides containing the p16INK4a fragment on the cell cycle. A - unsynchronized cell line - 293, B - synchronized in the S-phase cell line - 293. The concentration of peptides is 40 μM.

Figures 3 and 4 show the effect of pAntp chimeric peptides containing the p16INK4a fragment on the cell cycle. A - unsynchronized cell line - 549, B - synchronized cell line - 549. The concentration of peptides - 40 μm.

Figure 5 shows the effect of chimeric peptides containing the p16INK4a fragment on the change in the level of apoptosis in the unsynchronized and synchronized cell line A549, the concentration of peptides is 40 μM. Y axis -% of apoptotic bodies.

Figure 6 shows the effect of chimeric peptides containing the p16INK4a fragment on the change in the level of apoptosis in unsynchronized and synchronized cell line 293, the concentration of peptides is 40 μM. Y axis -% of apoptotic bodies.

7 shows a comparison of the cytotoxic effect of chimeric peptides pAntp_p16 (1, 2) on 293 cell culture.

Figure 8 shows a scan of the A549 cell line incubated for 15 minutes with the peptide using a LeicaTCS SP2 laser scanning microscope. The figure shows cells in which the chimeric peptide p16p_Antp-FITS is visible as luminous dots.

Figure 9 shows the distribution of pAntp_p16 protein in A549 cells over time. 1, 15 and 30 minutes after the addition of protein. Laser scanning microscope Leika.

Figure 10 shows the dynamics of the FITC accumulation of the labeled chimeric peptide p16p_Antp in the mononuclear fraction of blood cells. The arrow indicates the time of addition of the peptide to the flow cytometer cuvette.

11 shows the kinetics of the accumulation of FITC - labeled p16__pAntp peptide in peripheral blood lymphocytes in vitro. X axis - time in cu, Y axis - fluorescence intensity (cu).

12 shows the dynamics of FITC accumulation of the labeled chimeric peptide p16p_Antp using the Jurkat cell line as an example. The arrow indicates the time of addition of the chimeric peptide to the flow cytometer cuvette.

On Fig shows the dependence of the intensity of intracellular fluorescence and the concentration of the drug in the medium. The first peak reflects the fluorescence intensity when adding to the Raji cell culture a chimeric peptide Antp_p16 labeled with FITC at a concentration of 0.1 μmol, incubation with the peptide for 15 minutes in the dark; the second and third peaks are the change in the intensity of intracellular fluorescence with a change in the extracellular concentration of peptidan 1 and 10 μmol, respectively.

On Fig shows the dependence of the intensity of fluorescence of cells and extracellular concentration of the chimeric peptide.

On Fig shows the change in the tumor volume of the inoculated cells in athymic mice with the introduction of the chimeric internalized peptide Antp_p16 into the tumor. A549 cell culture was transplanted into mice and the Antp_p16 peptide was administered at doses of 0.1 and 0.2 mg.

On Fig shows the change in tumor volume of the inoculated cells in athymic mice with the introduction of the chimeric internalized peptide Antp_p16 into the tumor. The mice had perivitis of HCT-116 cells, the peptide was administered at a dose of 0.1 mg.

On Fig shows photographs of experimental animals at the time of 7 injection of the studied internalizable peptide P16_Antp. A and B are photographs of mice with transplanted A549 cells, A is a mouse from the control group on day 16 of the experiment, the tumor has a size of 6.0 × 7.0 mm. B - a mouse from the experimental group on day 16 of the experiment with the introduction of P16_Antp at a dose of 0.1 mg, the tumor size 3.0 × 3.0 mm Figures C and D are mice with transplanted HCT-116 cells, C is a mouse from the control group on day 18 after inoculation of tumor cells (tumor size 11.5 × 13.5 mm). D - mouse from the experimental group after 7 injections of P16_Antp at a dose of 0.1 mg, tumor 4.5 × 4.5 mm in size.

On Fig shows a two-parameter cytogram of breast cells. The X axis shows PI staining, the Y axis shows cytokeratin stain. Region - “cytokeratin-positive apoptosis” is indicated by an arrow.

On Fig shows the combined effect of the chemotherapeutic agent 5-fluorouracil at a concentration of 100 nmol and the chimeric peptide pAntp_p16 (2) at a concentration of 40 μmol. The distribution of the phases of the cell cycle of A549 cell culture after 24 hours of incubation together with the chemotherapy and the studied peptide.

The implementation of the invention

Materials and research methods.

1. Methods for producing chimeric peptides

1.1. Genetic engineering method for producing a chimeric peptide

Obtaining a genetic engineering construct encoding the chimeric p16-Antp peptide, the production and purification of the hybrid peptide was carried out according to the following methods. A DNA fragment encoding the transduction domain of the Antennapedia protein (Antp, 140-301 a, o.), From Drosophila melanogaster, produced by PCR (polymerase chain reaction) and a chemically synthesized fragment of the p16 protein gene (82-102 a.o.) was cloned into vector pQE16. Based on the obtained recombinant plasmids, the plasmid pR762 was constructed. pR762 contains a DNA region encoding the p16-Antp chimeric peptide, consisting of fragments of the p16 (82-102 a.o) and Antp (140-301 a.o) proteins. A block of 6 histidine residues is contained in the N-terminal part of the encoded peptide, which makes it possible to purify this protein using affinity chromatography on Ni-NTA agarose. The production of the chimeric peptide was carried out in the strain E. coli M15 [pREP4], capable of expressing this protein in significant quantities (about 5% of the total protein of the cell). The transformation of E. coli cells with plasmid pR762 with the p16-Antp fusion gene was performed by electroporation. Induction of chimeric protein synthesis and chromatographic purification on Ni-NTA agarose was carried out in accordance with the standard protocol of QUIAGEN. Additional purification was performed by high performance liquid chromatography (HPLC). The purity of the obtained product was checked by polymacrylamide gel electrophoresis according to Lemmley. The resulting peptide was sterilized by filtration through a filter with a pore size of 0.2 μm, stored at -70 ° C in 200 μl portions and used for further experiments.

1.2. Obtaining a chimeric peptide by the solid-phase method.

Some peptides used in the work were obtained by the solid phase method using the Boc / Bzl strategy and the following protective groups for the side functions of amino acids: Asp (cHex), Glu (cHex), Arg (Tos), Met (O), Ser (Bzl), Thr (Bzl), His (Bom), Lys (2Cl-Z) and Trp (CHO). A styrene-1% divinylbenzene copolymer containing 0.53 mmol / g Boc-Lys (2Cl-Z) -PAM manufactured by Advanced Chem Tech Inc., USA, amino acid derivatives, condensing agents and catalysts manufactured by the Peptide Institute was used as a polymer substrate Inc., Japan, all solvents and trifluoroacetic acid (TFA) are domestically produced. Boc groups were released by the action of 50% TFA in dichloromethane (DCM), followed by neutralization with a 5% solution of diisopropylethylamine (Fluka, Switzerland) in DCM. The syntheses were carried out in a flow reactor with continuous recording of changes in the volume of the peptidylpolymer (swellographic monitoring. Condensations were performed using TBTU (Bachem, Switzerland) in the presence of 1-hydroxybenzotriazole (HOBt) and N-methylmorpholine [59]. The completeness of the passage of the condensation reactions at each stage was monitored on a qualitative ninhydrin test. Four-fold excesses of activated derivatives were used during the first and, if necessary, re-condensations. In the synthesis of fluorescently labeled FITC-P analog The 16-Ant N-terminal residue was added in the form of Fmoc-Lys (Boc) -OH and, after removal of the Boc group / neutralization, a solution of 100 eq of fluorescein isothiocyanate in dimethylformamide (DMF) was added to the peptidyl polymer and the reaction was carried out for 20 hours. Fmoc and / or formyl groups that blocked the lateral functions of tryptophan residues were removed from the polymer substrate by treating the resin with a 20% solution of piperidine in DMF (0 °, 2 hours). Peptides were cleaved from the polymer substrate while the permanent protective groups were removed with liquid hydrogen fluoride in two stages according to the “low-high HF” procedure. After lyophilization, the peptides were converted to acetate by passing through a short column filled with Amberlite IRA-410 ion exchange resin (Serva, Germany) (3 g, acetate form), and they were separated from non-peptide byproducts by chromatography on a 2.6 cm × 70 cm column with Sephadex G -10 (Pharmacia, Sweden), elution of IN AcOH. The frontal peak was collected and lyophilized. For all peptides, adequate molecular weights were obtained on a VISION 2000 MALDI-TOF mass spectrometer (Thermo Bioanalysis Corp. UK): 4485 and 5002, respectively.

2. Studies of chimeric peptides

2.1. Investigation of the influence of the position and size of the peptide vector on the antiproliferative activity of chimeric peptides.

One of the main objectives of the research was to study the influence of the position of the functional group and the size of the peptide on their biological activity; for this, three p16_pAntp peptides (1-3) were synthesized (Table 1). Differences in the data of chimeric peptides at the location of the active center relative to the N-C ends of the molecule and the presence of an insert of 44 amino acids (SERKRGRQTYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLTE) for peptide 3 (Table 1). Peptides 1-3 consist of the functional part of p16INK4a and the internalizable pAntp sequence and differ in the location of the functional group relative to the N-, C-ends of the molecule and in the presence of the insert for peptide 3 (obtained by genetic engineering method).

The p16_pAntp (1-2) peptides differ in the location of the p16INK4a functional group: peptide 1 - p16INK4a is located at the C-terminus of the molecule, peptide 2 - p16INK4a is located at the N-terminus, and the peptides are obtained by solid-phase synthesis. In the case of the chimeric peptide p16_pAntp (3) obtained by genetic engineering, the functional group p16INK4a is located at the N-terminus, but there is an insert of 44 AKOs.

When studying the properties of these peptides, synchronized and non-synchronized cell cultures were also studied. The peptide was added to the synchronized culture at the moment the synchronization block was removed; to the non-synchronized one, at a monolayer density (for adhesive cultures) of ~ 50%.

The results on the change in the distribution of cells by the phases of the cell cycle for peptides pAntp-p16INK4a (1,2,3) are shown in Figures 1-4. The addition of the chimeric peptide (1) (Table 1) containing the p16INK4a fragment does not cause the expected cytostatic effect. As in the case of synchronized and unsynchronized culture, the distribution of cells in the phases of the cell cycle practically does not differ in comparison with the control. However, the addition of peptides 2 and 3 leads to a noticeable decrease in the S phase, while the number of cells in G0 / 1 also increases. This is more clearly seen in experiments on synchronized cultures.

The experiments conducted on cell line A549 (Figures 3 and 4), found similar changes in the cell cycle for all studied peptides.

The results indicate that the chimeric peptide pAntp-p16INK4a (1) does not have an antiproliferative effect on cells. While the addition of pAntp-p16INK4a chimeric peptides (2 and 3) has an antiproliferative effect on cells and leads to an increase in cells in the G1 phase.

The absence of a cytostatic effect when chimeric peptide 1 is added is probably due to a change in the conformation of the peptide molecule due to the location of the pAntp internalizing sequence from the N-terminus of the chimeric peptide (in the case of peptides 2 and 3, pAntp is located at the C-terminus).

At the next stage of the work, the dependence of the cytotoxic effect exerted by the studied chimeric peptides with the pAntp peptide vector was evaluated. An experiment to determine the level of apoptosis was performed on cell lines A549 (human, lung carcinoma), 293 (human, fetal kidney) and MCF-7 (human, breast adenocarcinoma). Cells were cultured according to standard methods in DMEM containing 10% PBS. A part of the cells was pre-synchronized using double thymidine block techniques and a synchronization technique using a depleted medium. The studied chimeric peptides p16p_Antp (1 and 2) were added to the culture medium. The cells treated with the pAntp_zam peptide were used as a negative control (the transport peptide (Antp peptide) was used as a control, or, in some experiments, the peptide in which amino acids V95 and V96 were replaced with alanine, which led to the loss of its functional activity). (Figures 5 and 6).

As can be seen from Figures 5 and 6, the addition of the control peptide pAntp_zam does not affect the level of apoptosis, while the addition of p16p_ntp (1, 2) significantly increases the number of cells entering apoptosis. Moreover, differences in the magnitude of the effect depending on the type of cells (for lines A549 and 293) were not found. A comparison of the effects of p16p_ntp 1 and 2 peptides on the apoptosis level for cell line 293 is shown in Figure 5. Figure 6 shows that the dependence of the number of apoptotic bodies on the concentration of p16_pAntp is a power-law. Comparison of the level of apoptosis with the addition of chimeric peptides showed that the level of apoptosis in the case of adding peptide 2 is almost 2 times higher than the level of apoptosis in the experiment with chimeric peptide 1. A more pronounced cytotoxic effect is observed on a synchronized culture. Thus, it was found that pAntp 1 and 2 chimeric peptides containing the p16INK4a fragment have pronounced cytotoxic effects in relation to cell cultures.

When comparing the cytotoxic and cytostatic effects of the chimeric peptides p16_pAntp 1 and 2, it can be concluded that a change in the position of the internalized pAntp sequence in the chimeric peptide from the C-terminus to the N-terminus leads to the disappearance of the cytostatic effect and a decrease in the cytotoxic effect.

The Figure 7 shows a comparison of the cytotoxic effect of chimeric peptides p16_pAntp (1, 2) on 293 cell culture.

Since the supposed intracellular effect of the studied peptides is the inhibition of cyclin kinases, a control peptide (Antp_zam) was synthesized containing a similar sequence from pl6INK4a protein (AKP 82-102) with 92 tyrosine replaced by 92 alanine. This replacement was chosen because earlier in Ferouse et al. it has been shown that substitutions of 91 or 92 AKOs result in the loss of the inhibitory ability of the peptide for the p16INK4a sequence.

Thus, a peptide comprising the cyclin D inhibitory sequence from p16 protein (20 amino acids) with the pAntp vector sequence (16 amino acids) located at the C-terminus of the functional amino acid sequence is most effective from the point of view of apoptosis activation.

2.2. The study of the penetration of internalizable peptides.

To record the penetration of peptides into the cell and to study the dynamics of accumulation, identical chimeric sequences conjugated with a fluorescent label fluorescein-isothiocyanate (FITC) were used. Because the inclusion of FITC molecules in the finished polypeptide product often leads to a significant change in its physicochemical properties and loss of physiological activity, therefore, a lysine molecule was attached to the original sequence at the N terminus, which carried one FITC molecule.

Using light fluorescence microscopy, it was shown that the studied peptides bind to cells and penetrate into Raji, Jurkatt, A549, 293 cell lines and human peripheral lymphocytes. Binding of the peptide to the cells and the absence of the effect of quenching fluorescence with trypan blue was shown by flow cytometry, which indicates the accumulation of the peptide inside the cell.

The most objective data on the accumulation of the peptide inside the cell were obtained using the method of scanning laser microscopy (Figure 8). Protein labeled with fluorescein isothiocyanate was dissolved in 0.9% NaCl. Cell lines A549, 293 were grown on sterile slides and placed in a special chamber directly under the microscope eyepieces. The medium was replaced with the medium with the studied protein. Protein saturation was observed over time. In addition, a layer-by-layer cell scan was performed to determine the localization of the peptide.

Analysis of the obtained images at this increase did not reveal the predominant location of the p16_pAntp protein in the cell. Obviously, the protein is distributed evenly in the cell compartments. The penetration of protein into the cell occurs quite quickly. And after 15 minutes of incubation, you can observe a relatively homogeneous distribution in the intracellular space (Figure 9).

The penetration rate of the peptide into the peripheral lymphocytes of human blood, as well as lymphocytes of cell lines (Raji, Jurkatt), was estimated using flow cytometry. This method allows you to study large concentrations of cells over time and is convenient for assessing the penetration rate of the studied peptide. Lymphocyte fluorescence was measured under the action of the p16-pAntp-FITC protein at pH 7.5 and pH 6.0. The principle of the method is based on a lower quantum yield of FITC fluorescence in solutions with a more acidic pH. Since the measurement speed is fast enough, the pH inside the cell cannot change. Lymphocytes were incubated with the peptide for 1-15 minutes, after which a 20-fold volume of phosphate buffer with pH 6.0 and pH 7.5 was immediately added to them. In this case, the difference in fluorescence intensity was estimated. In most measurements, the fluorescence intensities did not significantly differ after 15 minutes of incubation. Thus, it can be assumed that after 15 minutes of incubation, the peptide completely penetrates into the cell.

To study the rate of accumulation of the peptide inside the cell, flow cytometry was used. For this, the FITC-labeled peptide was introduced directly into the measuring tube of the flow cytometer, which made it possible to record the dynamics of changes in cell fluorescence (Figure 10). We studied the mononuclear fraction of white blood cells from healthy donors isolated on a ficoll gradient. The Figure 10 shows the kinetics of changes in the fluorescence of cells after adding the peptide labeled with FITC. It can be seen that after a small lag phase, the peptide rapidly accumulates in the cells of normal lymphocytes. The final concentration is achieved in a time equal to ~ 1 min.

Figure 11 shows only a fragment of a curve reflecting the kinetics of the accumulation of a chimeric protein in a cell. The obtained kinetics curve is better described by a power law

a function of type C = const1-const2 * t + const3, which reflects the regression coefficient (= 0.79).

A similar kinetics of peptide accumulation was also obtained for malignant lymphoma cells. As models, the Jurkat lines (originating from lymphoblastic leukemia and having a T-cell phenotype) and Raji - originating from Burkit's B-cell lymphoma were studied. Figure 12 shows the dynamics of the accumulation of FITC-labeled peptide in Jurkat cells.

The graph shows that the peptide penetrates into tumor cells with high speed. The time to reach maximum concentration is less than 1 min. The kinetics of accumulation was studied at room temperature (t = 20 ° C). It should be emphasized that the penetration mechanism of the studied class of peptides is not yet known. However, it has been shown that accumulation is equally effective even at a temperature of + 5 ° C and is not associated with energy expenditures in the cell, is not mediated by cellular receptors, and does not use the phage and pinocytosis pathways. The authors also confirmed the fact of accumulation of the synthesized peptide containing the internalizable fragment at different temperatures (up to + 5 ° C).

The dependence of the peptide accumulated in the cells on its extracellular concentration was also investigated. Figure 13 and 14 show the change in the fluorescence intensity of cells depending on extracellular concentration.

Figure 14 shows the same data illustrating the fact that the ratio of extra- and intracellular concentration is linear (in the studied concentration range). Because the studied peptides can cross the cell membrane in both directions, it can be assumed that the peptides accumulated in the cell can leave it with a decrease in extracellular concentration. Investigation of the processes of export of peptides from cells may be the subject of separate studies.

Thus, as a result of the experiments, it was shown that the kinetics of accumulation of the chimeric peptide containing the internalizable fragment and the protein fragment p16INK4a has a power-law character with a dependence of the type C = const1 + const2 * t ² . It was also shown that the dynamics of accumulation and intracellular distribution of the peptide in normal and tumor cells does not differ in nature and in the rate of accumulation.

2.3. Research materials

2.3.1. Cell line management

The study of the effect of peptides on proliferative activity was carried out in vitro on cell cultures. The following cell lines were used for this: Jurkat (T-lymphoblastic leukemia, provided by IBC, Russia), 293 (human, embryo kidney, transformed DNA of type 5 adenovirus (Ad5), provided by RAAS), A549 - (human, lung carcinoma, provided RAAS), Raji - (human, Burkit's lymphoma, provided by the Institute of Carcinogenesis), MCF-7 (human, breast cancer, provided by the IBC, Russia). Jurkat and Raji cells are adhesion cultures, 293, A549 and MCF-7 are suspension cultures. For management of Jurkat and Raji cells, RP Ml-1640 medium (PanEco, Russia) was used; for maintenance of 293, A549 and MCF-7 DMEM medium (PanEco, Russia). These cell lines differ in expression of the p16INK4a gene, as can be seen from Table 2, in the Jurkat, A549 and MCF-7 cell lines, p16INK4a is not expressed, and its expression is not disturbed in the Raji and 293 lines.

table 2 The expression level of p16 and p53 genes for cell lines used in the work Gene Cell line Jurkat Raji A549 Mcf7 293 Expression level P16 - + - - +

2.3.2. Suspension crop management (Raji, Jurkat)

Cells were cultured in RPMI-1640 medium (PanEco, Russia) containing 10% FSB (PanEco, Russia), 300 ng of the antibiotic-antimycotic gentamicin (PanEco, Russia) and 2 mM L-glutamine (PanEco, Russia) (37 ° С, 5% CO2 atmosphere) (incubator). Upon reaching a high density, the cells were washed from the old medium by centrifugation for 2 min at 1200 rpm./min, resuspended in RPMI-1640 medium containing 10% FSB, and reseeded.

2.3.3. Maintaining adhesive cultures (293, A549, MCF-7)

Cells were cultured in DMEM (PanEco, Russia) containing 10% FSB, 300 ng of antibiotic antimycotic and gentamicin and 2 mM L-glutamine (37 ° C, 5% CO ₂ atmosphere). Supernatant was removed from the plate to remove and seed cells. Washed with a solution of phosphate-buffered saline (PBS). 0.25% trypsin (PanEco, Russia) was added to EDTA, the cells left in 5 minutes (sometimes it was necessary to incubate at 37 ° C in 5% CO ₂ atmosphere for 10 minutes). Cells were selected, washed from trypsin by centrifugation for 2 min at 1200 rpm, resuspended in DMEM medium containing 10% FSB, and the vial was placed in an incubator at 37 ° C in 5% CO ₂ atmosphere.

When working with cell cultures, the necessary requirements for the sterility of the reagents and devices used were observed. Cells were pre-cultured (37 ° C, 5% COz atmosphere) in culture medium containing 10% FSB, or synchronized if necessary.

2.3.4. The study of the antitumor activity of the chimeric peptide p16_Antp in vivo (local administration)

In nude mice (Nude), the antitumor activity of the chimeric internalized peptide P16_Antp was studied.

Research Methodology

Mice were inoculated with tumor cultures of human cells of lines A549 and HCT-116 in an amount of about 1 million cells per mouse.

A549 cells were inoculated into 39 mice, of which 20 mice made up the control group, they were subsequently given a placebo. 10 mice made up 1 experimental group. After the formation of a palpable tumor (4 days after transplantation of cells), they began to inject the studied peptide at a dose of 0.1 mg directly into the tumor. Nine mice constituted the 2nd experimental group; after the formation of the tumor node, on the 4th day of inoculation, 0.2 mg of the peptide was also introduced into the tumor bed. The peptide in the experimental groups was administered once every two days. The experiment lasted 24 days.

results

In the experimental groups, a decrease in the growth rate of tumors was observed, and on the 6th day after the start of the administration of the peptide, a noticeable decrease in the volume of tumors in the experimental groups was observed compared to the control (Figure 15). The experiment lasted 24 days. During this time, 10 injections of the chimeric peptide were carried out in the experimental groups. In the control group, on the 24th day of the experiment, the average tumor volume in mice was 95.24 mm ³ ; one mouse died. In the first experimental group (the dose of the administered peptide is 0.1 mg), the average tumor volume was 39.29 mm ³ , one mouse died. In the second experimental group (the dose of the administered peptide is 0.2 mg), the average tumor volume was 57.51 mm ³ , but there were no dead animals.

When mice were injected with transplantable human culture NST-116, the animals were divided into two groups: the control group consisted of 10 mice to which the test peptide was not subsequently administered, and the experimental group (10 mice) to which the test chimeric peptide P16_Antp was administered at a dose of 0.1 mg to tumor node. The experiment lasted 28 days, 11 injections of the peptide were made in the experimental group, the first injection was made on day 6 after the transplantation of tumor cells. The tumor node from HCT-116 cells was large and ulcerated. In the control group, on the 28th day of the experiment, the average tumor volume was 679 mm ³ ; 3 mice died during the experiment. In the experimental group, a lower tumor growth rate was observed (Figure 16), on the 28th day of the experiment, the average tumor volume was 225.4 mm ³ , there were 2 dead animals in the experimental group.

The Figure 17 presents a pair of mice from different groups after 7 injections of the studied peptide. A noticeable decrease in the tumor node in the experimental groups is visible.

findings

Local administration of the chimeric peptide p16-Antp leads to significant (more than 50%) inhibition of the growth of experimental models of human tumors (breast cancer, colorectal cancer).

2.3.5. The study of the cytotoxic properties of chimeric peptides in short-term cultures of human tumors

We studied the antitumor activity of chimeric peptides, including the internalized Antp fragment and the functional fragments of cyclin kinase inhibitors p16INK4a (DAAREGFLDTLVVLHRAGAR-S-RQIKIWFQNRRMKWKK (SEQ ID N0.3)). 150 mg of the p16-Antp peptide were synthesized by the solid-phase synthesis method (see the methods section). To study the effects on human tumors, the short-term culture method was used.

A technique for producing short-term tumor cultures from surgical material.

Short-term cultures were obtained from operational material. The site for the preparation of culture was taken as soon as possible after the operation and with the participation of the pathologist. A section of tissue was cut out, an average of 1.5 cm ³ . The tissue was mechanically crushed, the cell suspension was placed in RPMI medium containing 5% PBS. After 24 hours of incubation at 37 ° C and 5% CO _{2, the} medium was replaced with a medium containing a chimeric peptide (p16_Antp at a concentration of 40 μM), or for a series of experiments on Taxol at a concentration of 100 nM or 500 nM containing both the peptide and Taxol. In control samples, the medium was replaced with fresh. The first point was shot - 0 hours.

Incubation took place at 37 ° C and 5% CO ₂ 24 hours, for a number of experiments 24 and 48 hours. The results were evaluated by flow cytometry (see in vitro methods for the method) using double staining of AnnexinV-PI samples and staining of fixed PI material. Assessed the level of particles positive for AnnexinV (early apoptosis), the level of particles positive for double label AnnexinV-PI (late apoptosis), the number of particles in unfixed samples that can include PI (necrosis); cell distribution according to the phases of the cell cycle, the level of the subdiploid peak (the number of particles with fragmented DNA, apoptosis). For the detection of apoptosis of epithelial cells, a double staining of the anti-cytokeratin-FITC-PI antibody was used. (Figure 18).

A total of 126 tissue samples were analyzed: of which 63 samples of pathological tissues (and 63 samples of normal tissues as controls) Of the 63 pathological samples, 47 malignant tumors were examined (10 - breast cancer, 15 - kidney cancer, 6 uterine cancer, 4 cancer prostate gland, 3 ovarian and lung cancers, and 2 cases of stomach cancer, pancreatic cancer, bladder cancer each.) In addition, tissue samples with fibroadenomas (N = 9), as well as 7 breast tissue samples with simple ductal hyperplasia were examined.

Apoptosis levels were analyzed 24 and 48 hours after the addition of peptides at a concentration of 40 μM. Some experiments were performed to study the combined cytotoxic effect of traditional chemotherapeutic drugs and the p16_Antp peptide on tumor cells.

2.3.6. The study of the antitumor activity of the peptide p16_Antp

Summary results on the cytotoxic activity of p16_Antp peptide are shown in Table 3.

Table 3 The average level of induced apoptosis in short-term tumor cultures when exposed to p16 Antp peptide (24 hours, 40 μM). Cancer type Induced Apotosis Level Mammary cancer 25.8 Kidney cancer 26.0 Cervical cancer 5.2 Pancreas cancer 18.9 Lung cancer 13.5 Stomach cancer 35,4 Prostate cancer 15.4 Bladder cancer 24.7 Ovarian cancer 12.3

An analysis of the results shows that the most sensitive forms of cancer for the cytotoxic effect of p16_Antp peptide are: cancer of the kidney, breast, stomach, and bladder. It should be noted that short-term bladder cancer cultures are characterized by a very high level of spontaneous apoptosis (up to 60-70%). Despite the fact that the addition of the peptide increases this level slightly, against this background, the results obtained may be non-representative.

2.3.7. The study of the cytotoxic effect on breast fibroadenoma cells

Interesting results were obtained in the study of tissue samples of breast fibroadenoma. In short-term cultures of these cells with a low level of spontaneous apoptosis (maximum up to 20%, average 12.8%), the peptide p16 exerted a significant cytotoxic effect and caused pronounced apoptosis after 24 hours with an average level of 38.2% with a maximum of 80%. A less pronounced cytotoxic effect was found for breast cells with signs of simple ductal hyperplasia (average level of induced apoptosis of 14.5%). These results are consistent with the data obtained by the author on the study of the ratio of proliferation activity and the level of spontaneous apoptosis in the pathological tissues of the mammary gland. It was shown that the ratio of proliferation and spontaneous apoptosis is highest for fibroadenoma tissue, and breast cancer cells are in second place in this indicator, followed by simple ductal hyperplasia and normal breast tissue. If one of the factors determining the sensitivity of cells to the cytotoxic effect of peptides containing proliferation inhibitors is the activity of cell division, then the sensitivity of fibroadenoma cells to the p16_Antp peptide is quite logical. The detected sensitivity effect of benign proliferative processes in breast tissue may be promising for their treatment. The results on the local introduction of peptides for the treatment of transplantable solid tumors suggest their effectiveness when introduced locally into the area of benign proliferative processes in the tissues of the mammary gland.

findings

The results obtained revealed a spectrum of human tumors sensitive to the p16_Antp peptide. It was found that localizations such as breast cancer, colorectal cancer, stomach cancer, bladder cancer, and kidney cancer are promising objects for further study of the cytotoxic (antitumor) effect of the studied peptide. The results are new, because in the available literature there is no data on the study of the effect of the analyzed peptide on human tumors.

2.3.8. Investigation of the combined effect of taxol / 5-fluorouracil / etoposide and p16_Antp peptide.

In medical practice, in the treatment of malignant neoplasms, chemotherapy drugs - cytostatics - are widely used. Their effect is based on the fact that cells under their influence stop at a certain phase of the cell cycle, and the combined administration of a drug with cytotoxic activity at this phase of the cycle allows the death of a large number of cells.

The studied chimeric peptides have been shown to have cytostatic and cytotoxic activity. Moreover, the cytostatic activity is specific and is the result of the direct effect of the peptide on the cell genome. It was proposed to investigate the combined effect of internalizable peptides and chemotherapeutic agents (Fig. 19).

The following chemotherapeutic drugs were studied that are used in medical practice and affect the cell cycle: Taxol, which causes cell retention in the G2 / M phase, Etoposide - delay in the S phase and 5-fluorouracil - delay in the G0 / G1 phase of the cell cycle. The most informative results with the combined action of chemotherapeutic agents and chimeric peptides were obtained with the use of the p16_pAntp peptide (2). In preliminary experiments on cell lines 293 and A549, the expected effects of a delay in the cell cycle in the phases characteristic of each studied drug were obtained and the optimal concentration was selected that allows one to observe the effect without causing total cell death. This concentration for all preparations was 100 nMol.

When studying the combined effects of chemotherapeutic agents and the chimeric peptide p16_ pAntp (2), a synergistic increase in the cytostatic effect was observed for the preparations of 5-fluorouracil and taxol, exceeding the additive therapeutic effect that could be expected in this case.

Claims (4)

1. The use of a chimeric peptide containing a functional 20-amino acid sequence from a protein inhibitor of cyclin kinases p16INK4a (SEQ ID NO.1) and a transport 16-amino acid sequence from an Antp protein (SEQ ID NO.2) for the treatment of a cancer selected from the group including colorectal cancer, kidney cancer, lung cancer, breast cancer, bladder cancer, pancreatic cancer, uterine cancer, prostate cancer, stomach cancer and ovarian cancer, while the amino acid transport sequence is attached to the end of the indicated functional sequence through group X, where X is an amino acid sequence containing from 1 to 50 amino acid residues. 2. The use according to claim 1, characterized in that the chimeric peptide has the amino acid SEQ ID NO.3. 3. The use according to claim 1, characterized in that the chimeric peptide has the amino acid SEQ ID NO: 4. 4. The use of a chimeric peptide containing a functional 20-amino acid sequence from a p16INK4a cyclin kinase inhibitor protein (SEQ ID NO.1) and a 16-amino acid transport sequence from Antp protein (SEQ ID NO.2) to create a pharmaceutical composition for treatment a cancer selected from the group consisting of colorectal cancer, kidney cancer, lung cancer, breast cancer, bladder cancer, pancreatic cancer, uterine cancer, prostate cancer, stomach cancer, and ovarian cancer, while an orthic amino acid sequence is attached to the C-terminus of the indicated functional sequence by group X, where X is an amino acid sequence containing from 1 to 50 amino acid residues.

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