RU2369402C1 - Chimeric peptide for epithelial and mesenchymal malignant neoplasms treatment - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicines, particularly to the use of chimeric peptide VP-22_p16INK4a for epithelial and mesenchymal malignant neoplasms treatment. The claimed chimeric peptide VP-22_p16INK4a contains two amino acid sequences. The first sequence comprises inhibitor of cycline kinases as active fragment p16INK4a as therapeutic agent and the second sequence comprises peptide VP22 of herpes simplex virus as carrier agent to deliver cycline kinase inhibitor into target cells.

EFFECT: enlarging the application range of medicine.

9 dwg

Description

The invention relates to medicines, namely to chimeric peptides containing a transport (internalizable, in the world literature there is also the term "cell penetrating peptides" - CPP) and functional part, intended for the treatment of epithelial and mesenchymal malignant neoplasms (i.e. non-lymphoid malignancies origin).

The creation of chimeric peptides with cytostatic and cytotoxic activity is a promising direction in the development of modern therapy for non-lymphoid malignancies.

From the description of the patent RU 2297241 C2 for the invention "Chimeric protein for the treatment of malignant lymphomas" (published on 04/20/2007) it is known that one of the directions for the treatment of tumor diseases is the use of cyclin kinase inhibitors, for example, proteins of the INK4a family that affect the cell cycle. It is also known that it is desirable that molecular constructs are delivered to the cell “targeted” and act on a particular intracellular signal or function. It is also known from the aforementioned document that, depending on the structure, different peptides can accumulate differently in different cell compartments, which made it possible to construct sequences with targeted delivery to different cell organelles.

The chimeric peptide VP-22_p16INK4a according to the patent RU 2297241 C2 has an increased biological effect due to the improved properties of the transport agent and is intended for the treatment of malignant lymphomas, however, it is not known about the possibility of its use for the treatment of epithelial and mesenchymal malignancies.

The objective of the present invention is to expand the field of use of the chimeric peptide VP-22_p16INK4a and the use of this peptide for the treatment of epithelial and mesenchymal malignancies.

The technical result of the invention is a biomedical effect, objectively manifested when using this chimeric peptide and consisting in solving this problem.

The technical result is achieved due to the use for the treatment of tumors of non-lymphoid origin of the chimeric peptide VP-22_p16INK4a containing two amino acid sequences, the first of which includes a cyclin kinase inhibitor in the form of the active fragment p16INK4a (amino acid residues 84-103 or 84-106) as a therapeutic agent and the second includes the peptide VP22 (amino acid residues 140-301) of the herpes simplex virus as a transport agent for transferring the cyclin kinase inhibitor into the target cells.

The chimeric peptide VP-22_p16INK4a contains the following amino acid sequence: DAATATRGRSAASRPTERPRA-PARSASRPRRPVE- DAAREGFLDTLVVLHRAGAR (hereinafter - SEQ ID NO. 1, the sequence from p16 is underlined).

Monitoring and synchronization of events in the process of cell division is carried out by a large complex of molecules, one of the key participants in this process are cyclins that activate the so-called cyclin-dependent kinases (CDKs). During the cell cycle, sequential activation of transcription of a specific cyclin occurs, followed by the formation of the active cyclin-CDK complex. Stop cell division at control (restriction) points (G1, S, G2) by inhibiting the corresponding cyclin complex through specific proteins. The cyclin D inhibitory protein family (G1-S transition control) is an INK4a peptide.

The p16INK4a peptide is the most studied of the indicated group. This peptide inhibits the activity of CDK4 and CDK6 complexed with cyclin D, which interferes with the phosphorylation of pRb and the release of E2F, and leads to a cell cycle arrest at the G1-S transition boundary. Structurally functional studies of this protein revealed the active fragment p16INK4a (amino acids 84-103 or 84-106), which is responsible for the inhibition of CDK4 and CDK6, which are complex with cyclin D.

The p16INK4a peptide plays an important role in the process of cell differentiation and aging, in addition, this peptide is not functional or absent in many tumor tissues. For malignant neoplasms of non-lymphoid origin, dysfunction of the p16INK4a peptide and overexpression of cyclin D are characteristic. Thus, in malignant neoplasms of non-lymphoid origin, there are impaired control of the restriction point R1 (G1-S transition).

As indicated in the description of patent RU 2297241 C2, the ability to use natural cyclin kinase inhibitors to control proliferation arose after the discovery of the ability of some proteins to transactivate. That is, the proteins synthesized in one cell could leave it and, penetrating into another cell, lead to the activation of certain genes. The study of the structure of these proteins revealed short (from 10 to 30 amino acids) amino acid sequences responsible for intracellular transport. It was shown that the addition of such a sequence to the structure of an arbitrary protein gives it the properties of intracellular and intranuclear internalization. Currently, about 30 sequences of such peptides are known.

The designed chimeric peptide molecule, including VP22 protein fragments and a fragment of p16INK4a protein, an inhibitor of cyclin kinases, was previously used exclusively for the treatment of malignant lymphomas. However, at present, there is experimental evidence of the suitability of using the chimeric peptide VP-22_p16INK4a for the treatment of epithelial and mesenchymal malignancies.

The possibility of carrying out the invention with the implementation of this purpose is confirmed by the following research results.

Materials and methods. Examples

Work with cell cultures

Cells were cultured at 37 ° C in 5% CO2 atmosphere in DMEM (PanEco, Russia) containing 10% fetal bovine serum (FSB) (PanEco, Russia), 10 μg / ml gentamicin antibiotic-antimycotic (PanEco, Russia) and 2 mM L-Glutamine (PanEco, Russia).

The chimeric peptide and antitumor preparations were dissolved in the culture medium and added to the cells upon changing the medium. The effect was evaluated after 24 hours of incubation.

Short-term human tumor cultures

To assess the effectiveness of the cytotoxic effect of the chimeric peptide VP-22_p16INK4a on human tumors, short-term cultures obtained from surgical material for breast cancer, stomach cancer, uterine cancer, and kidney cancer were used. The effect of the chimeric peptide on short-term skin cultures, unchanged gastric mucosa, unchanged endometrium, and unchanged kidney was also evaluated. Short-term cultures were obtained by mechanical disaggregation of the surgical material, cells were incubated for 24 hours at 37 ° C in 5% CO ₂ atmosphere in RPMI medium (PanEco, Russia) containing 5% fetal bovine serum (FSB) (PanEco, Russia) and necessary for growth components. Then, the medium was replaced with a medium containing the chimeric peptide VP-22_p16INK4a at a concentration of 40 μM. The effect was evaluated after 24 and 48 hours of incubation with the peptide.

When working in vitro, the effects of chimeric peptides on cells were evaluated using flow cytometry (DAKO Galaxy flow cytometer). We studied the apoptosis level, the number of dead cells by AnnexinV-PI staining (propidium iodide), and the quantitative distribution of cells by phase of the cell cycle by PI staining.

Coloring on AnnexinV. The AnnexinV KIT kit (Caltag laboratories LI 2004) was used. Cells were washed from the medium in cold phosphate buffered saline (PBS), resuspended in 1 × Binding Buffer at a concentration of 106 cells per ml. 100 μl of the resulting suspension were taken and 5 μl of AnnexinV-FITC and 10 μl of PI were added to it. Incubated in the dark at room temperature for 15 minutes. 400 μl of 1 × Binding Buffer was added and analyzed on a flow cytometer.

Coloring PI. Cells were washed from serum by double centrifugation in 1 ml of PBS (4 minutes at 2000 rpm). The supernantant was removed, 300 μl of PBS was added, 700 μl of ice-cold 70% ethyl alcohol was added dropwise and left at -20 ° C overnight (fixation). Fixed cell samples were besieged by centrifugation (4 minutes at 2000 rpm), washed with 1 ml of PBS and centrifugation was repeated. To the precipitated cells were added 100 μl of PBS and 20 μl of RNase A (1 mg / ml) and incubated for 30 minutes at 37 ° C. Next, 30 μl of PI was added to the suspension and incubated for 40 minutes at room temperature in the dark. At the end of the incubation, the volume of the test sample was adjusted to 1 ml with PBS.

In the study of chimeric peptides in vivo, the antitumor effect was evaluated on nude mice (nude) with transplanted human tumor cells of lines A549 (lung cancer) and HCT-116 (human colon cancer).

To study the effects on human tumors, the short-term culture method was used.

Study of the effect of a chimeric peptide on short-term cultures of breast cancer, stomach cancer, and kidney cancer. It was shown that for this model, the most sensitive variant of the tumor is kidney cancer.

In vivo experiments

In nude mice (Nude), the antitumor activity of the chimeric internalized peptide VP-22_p16INK4a was studied. Tumor cultures of human cells of lines A549 (lung cancer) and HCT-116 (colon cancer) in the amount of about 1 million cells per mouse were subcutaneously transplanted into mice. The introduction of the peptide began after the detection of the tumor node (3-4 mm) on 4-6 days after inoculation of the tumor. 100 (first group) or 200 μg (second group) of the peptide was administered every other day. Both tumor lines received 50% inhibition of tumor growth. There were no differences in effect for doses of 100 and 200 mcg.

The results of studies of the treatment of malignant neoplasms of non-lymphoid origin using the chimeric peptide VP-22_p16INK4a are illustrated by the following drawings:

Figure 1: the level of apoptosis in short-term cultures of unchanged renal tissue (normal) and kidney cancer (cancer) when incubated with the chimeric peptide VP-22_p16INK4a at a concentration of 40 μm for 24 hours. Average values for 4 experiments. The number of apoptotic particles was determined as positive according to AnnexinV with double staining of AnnexinV-PI;

Figure 2: cytotoxic effect of the chimeric peptide VP-22_p16INK4a on short-term cultures of unchanged renal tissue (normal) and kidney cancer (cancer). The average number of particles that are positive according to AnnexinV, and particles including PI, with double staining of AnnexinV-PI according to four experiments. Incubation with a chimeric peptide for 24 hours;

Figure 3: the level of apoptosis in short-term cultures of unchanged tissue of the gastric mucosa (normal) and stomach cancer (cancer) when incubated with the chimeric peptide VP-22_p16INK4a at a concentration of 40 μm for 24 hours. Average values for 4 experiments. The number of apoptotic particles was determined as positive according to AnnexinV with double staining of AnnexinV-PI;

Figure 4: cytotoxic effect of the chimeric peptide VP-22_p16INK4a on short-term cultures of unchanged tissue of the gastric mucosa (normal) and gastric cancer (cancer). The average number of particles that are positive according to AnnexinV, and particles including PI, with double staining of AnnexinV-PI according to four experiments. Incubation with a chimeric peptide for 24 hours;

Figure 5: apoptosis level in short-term cultures of unchanged uterine body tissue (normal) and cervical cancer (cancer) when incubated with the chimeric peptide VP-22_p16INK4a at a concentration of 40 μM for 24 hours. Average values for 4 experiments. The number of apoptotic particles was determined as positive according to AnnexinV with double staining of AnnexinV-PI;

6: cytotoxic effect of the chimeric peptide VP-22_p16INK4a on short-term cultures of unchanged uterine tissue (normal) and cervical cancer (cancer). The average number of particles that are positive according to AnnexinV, and particles including PI, with double staining of AnnexinV-PI according to four experiments. Incubation with a chimeric peptide for 24 hours;

Fig. 7: apoptosis level in short-term cultures of unchanged breast skin tissue (normal) and breast cancer (cancer) when incubated with VP-22_p16INK4a chimeric peptide at a concentration of 40 μM for 24 hours. Average values for 4 experiments. The number of apoptotic particles was determined as positive according to AnnexinV with double staining of AnnexinV-PI;

Figure 8: Cytotoxic effect of the VP-22_p16INK4a chimeric peptide on short-term cultures of unchanged breast tissue (normal) and breast cancer (cancer). The average number of particles that are positive according to AnnexinV, and particles including PI, with double staining of AnnexinV-PI according to four experiments. Incubation with a chimeric peptide for 24 hours;

Figure 9: volume change of the tumor node in nude mice. 1 mln cells of HCT-116 (human rectal cancer) were subcutaneously transplanted into the mice. The chimeric peptide VP-22_p16INK4a at a dose of 0.1 mg was administered to the experimental group (experiment) in the area of the tumor node, the control group (control) was injected into the area of the tumor node saline. The first injection was made on day 6 after transplantation of HCT-116 cells.

The results of the experiments shown in figures 1-9, which show an increase in the level of apoptosis and necrosis in malignant neoplasms of non-lymphoid origin using the VP-22_p16INK4a chimeric peptide, confirm the pronounced cytostatic and cytotoxic effect in relation to tumors of non-lymphoid origin both in vitro and in animal models.

The described experiments on the intratumoral administration of peptides in the subcutaneous version of tumor inoculation were carried out the first in the world. The literature does not describe similar experiments.

Moreover, the results on short-term models of human tumors were obtained for the first time in the world. It was shown for the first time that a peptide with the structure VP-22_p16INK4a can be used to treat malignant neoplasms of non-lymphoid origin.

Claims (1)

The use of the chimeric peptide VP-22_p16INK4a containing the amino acid sequence of SEQ ID NO.1 for the treatment of epithelial and mesenchymal malignancies.

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