RU2490028C1 - Method of treating oncological diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely oncology, and may be used for the purpose of the integrated treatment of the patients with II-IV breast cancer. That is ensured by administering cytostatic preparations after the one-stage chemotherapy, including: cyclophosphan (500 mg/m²) and doxorubicin (50 mg/m²) (the schedule AC) or cyclophosphan (500 mg/m²), doxorubicin (50 mg/m²) and fluorouracil (500 mg/m²) (the schedule FAC) intravenously. Besides, the preparation Panagen in the form of gastroenteric-coated tablets 30 mg/day is regularly divided over an active period of day that makes one tablet of the preparation Panagen for six times every two hours; the administration starts on the third day following the chemotherapy. The length of the administration of the preparation Panagen is 17 days and continued until the chemotherapeutic courses according to the above schedules completed.

EFFECT: method enables the higher clinical effectiveness in breast cancer that ensures preserving and stimulating granulocyte-macrophage and lymphocyte hematopoietic lineages that makes compensation for the negative effect of leukoreducing effect of the cytostatic preparations.

1 cl, 40 dwg

Description

The invention relates to medicine and can be used in the complex treatment of patients with stage II-IV breast cancer with preparations of the cytostatic group, in particular, a combination of the anthracycline cytostatics of doxorubicin and cyclophosphamide (AC scheme), as well as with the addition of the third cytostatic fluorouracil scheme ( FAC scheme).

A known treatment method based on the use of mono-preparations of the cytostatic group, for example, cisplatin for the treatment of patients with squamous cell carcinoma of the trunk of the body, by using cisplatin at a dose of 100 mg / m ² (I day), intravenously drip 30 mg / m ² once a week, or 60-150 mg / m ² every 3-5 weeks, or 20 mg / m ² daily for 5 days, repeated every 4 weeks, or 50 mg / m on the 1st and 8th days every 4 weeks, drug mitomycin C for the treatment of patients with cancer of the stomach, intestines, pancreas, bladder, breast, lung, vulva, pres atelnoy gland (consisting of combinations of drugs), by using mitomycin C intravenously at a dose of 10 mg / m ² 1 times in 3-4 weeks (consisting of combinations of drugs) or as 2 mg / ^m2 from the 1st to 5th and from the 8th to the 12th days of the course, the total dose of 50 mg / m ² , or of dactinomycin for the treatment of patients with uterine choriocarcinoma, soft tissue sarcoma, malignant tumors of the testis, melanoma, lymphosarcoma, neuroblastoma using intravenous dactinomycin 350 μg / m ² daily for 5 days every 3-5 weeks or 1-2 times a week for 3-5 Ned b, total dose 3000 mcg [1].

The method is characterized by relatively low treatment efficiency and limited use, since according to this method, not all types of neoplastic tissue are equally sensitive to the action of a cytostatic agent in the form of a single drug.

There is also a method based on the use of high-dose chemotherapy (XT), which includes continuous infusion of 5-fluorouracil 200 mg / m ² daily for a long time, epirubicin 50 mg / m ² and cisplatin 60 mg / m ² every 3 weeks for 6 months [2, 3].

The disadvantage of this method is the relatively low effectiveness of the treatment.

In addition to the above, a known method of treatment with epirubicin 100 mg / m ² and cyclophosphamide 400 mg / m ² with support for G-CSF with a 14-day interval. An objective response was received in 96.5% of cases (4 complete and 25 partial remissions). 36 patients received intensive treatment (T2> 3.5 cm, T3No) using epirubicin 65 mg / m, cyclophosphamide 600 mg / m and 5-fluorouracil 600 mg / m ² on day 1, with the support of G-CSF 5 μg / kg , days 7 to 12. The intervals between courses were 14 days. After three courses, the clinical effect was obtained in 100% of cases; 80.5% of patients underwent surgery [4, 5].

This method has limited applicability and relatively low efficiency.

Known is a method for treating stage IIIA and IIIB breast cancer with doxorubicin 90 mg / m ² and cyclophosphamide 1000 mg / m ² with the support of G-CSF with a 3-week interval. The overall clinical effect was 98%, complete clinical regression - 50%, complete histological regression - 14%. In 40 patients receiving 5-fluorouracil 750 mg / m ² (days 1-4), epirubicin 35 mg / m ² (days 2-4), cyclophosphamide 400 mg / m ² (days 2-4) with an interval of 3 weeks, with G-CSF support, clinical complete regression was 37.5%, partial - 45%, complete histological remission was observed in 15% of patients [6].

This method also has limited applicability and relatively low efficiency, since it does not activate the anti-cancer immunity of the body and only slightly increases the relapse-free interval in patients with breast cancer. In patients with locally advanced disease, 5-year relapse-free survival is from 13% to 45%, and 10-year-old from 10% to 30%. Metastatic breast cancer remains an incurable disease. For 2 and 5 years from the time of diagnosis, an average of 30% and 5% live, respectively [7, 8].

In addition, it is known that all methods using cytostatics have an extremely negative effect on hematopoiesis.

Also known are methods of treatment based on the action of a combination of cytostatic drugs and subsequent immunotherapy. So known drug Ingaron, which is an interferon-gamma. By its properties, it is a unique natural cytokine that provides the formation and stimulation of cellular immunity, which shows the feasibility of using gamma interferon in combination with XT in a number of malignant tumors. The combination of XT with immunotropic substances can significantly reduce the toxic and immunosuppressive effect of antitumor drugs. [9].

A known treatment method based on the use of cytostatic drugs followed by immunotherapy with specific oligonucleotides with a phosphorothioate backbone and a specific set of nucleotides (CpG) [10].

However, this method also has a low treatment efficiency due to the fact that immunomodulating CpG nucleotides act mainly on antigen-presenting dendritic cells (DCs), activating their maturation and specific activities through the TLR receptor system, and are not able to activate DCs through other molecular activation pathways So recently, much attention has been paid to the study of sensory pathways involved in the activation of DCs with drugs of linear double-stranded DNA, which are not associated with the functioning of TLR tori. The main metabolic pathways that detect and transduce a signal determined by the interaction of DCs and exogenous DNA molecules are described. These include the NRL, STING dependent pathways, as well as the signaling pathway mediated by the activity of RNA polymerase III [11-13] The completion of the signal transduction process by any of these pathways is the activation of transcription factors nfκb, API, groups of IRF proteins that are internalized in the nucleus and bind and activate IFN- (3 promoter. IFN-β is secreted, binds to the receptor (IFNAR) and activates the metabolic pathway for the production of a large number of interferon-inducible genes via the Jak / STAT autocrine or paracrine mechanism [14, 15]. As a result of the decree GOVERNMENTAL DC processes have increased expression of surface molecules MHCI, MNSP, costimulatory molecules (CD40, CD80, CD86), chemokine receptors. DCs to secrete chemokines and cytokines in order to attract effector cells and their differentiation [16].

The main disadvantage of these methods of treating cancer is their limited effect on body systems that induce anti-cancer adaptive immunity in the case of using cytostatics as a single drug, and high toxicity and inhibitory effects on leukopoiesis in treatment methods using cytostatics in combination with immunotherapeutic drugs.

To partially address these shortcomings, a rather extensive arsenal of drugs used for correcting hematopoiesis therapy after cytoreduction therapy with cytostatics has now been developed and used. Two principal types of drugs are used, one of which induce the mobilization of blood stem cells (CCM) and the proliferation of late precursors of the neutrophilic hematopoietic germ, and drugs that induce proliferation of the precursors of all hematopoietic germs of varying degrees of commit. The first group includes drugs based on colony-stimulating factors (G-CSF and GM-CSF), the second group includes drugs based on exogenous DNA.

Preparations based on fragmented exogenous DNA have an activating proliferation effect on all types of hematopoietic precursors to varying degrees of their maturity. This action allows you to simultaneously start the division of the CCM, increasing the percentage of dividing cells several times. Such an action, as well as the mobilization of CMC under the influence of colonies - stimulating factors, leads to the appearance in the peripheral blood of the whole gamut of uniform elements of the leukocyte and lymphocytic rows. However, the release of mature cells into the bloodstream as a result of such an effect, unlike mobilization, which is a forced exit of CCMs and their committed descendants from stromal niches, is physiological and therefore is delayed by 1-3 days. The use of fragmented DNA-based drugs for the prevention of myelosuppression and stimulation of hemo- and leukopoiesis in cancer patients is a generally accepted approach, which has its positive aspects, since its use does not interfere with the functioning of the basic cellular systems of the body.

In this regard, it is possible to indicate the popularity of the treatment method with the use of the “Deoxinat” preparation, which is the sodium salt of double-stranded deoxyribonucleic acid, obtained from sturgeon milk and partially depolymerized by ultrasound to a molecular weight of 2.7-4.0 × 10 ⁵ Yes . The DNA content in the preparation is more than 90%, RNA (from 3 to 5%) and non-histone type proteins associated with DNA are present as impurities in the preparation. The drug has an antitoxic and radioprotective effect and is used as a stimulant of leukopoiesis in chemoradiotherapy conducted in cancer patients [17].

However, “Deoxynat” does not have its own antitumor activity and therefore is used only as an auxiliary leukostimulating drug for the prevention of myelodepressions during chemoradiotherapy of cancer patients, while the leukostimulating effect develops slowly and persistent changes are observed after 4-10 days. The radioprotective effect of “Deoxynat” is indirect in nature and is aimed at correcting leukopenia and stimulating the immune system that develop in acute radiation sickness of the II-III degree of severity [18].

A known method of stimulating leukopoiesis in patients with advanced forms of tumors of the abdominal cavity by a single subcutaneous injection of exogenous DNA obtained from sturgeon milk. DNA is presented in the form of a low-polymer powder containing at least 80% of the native sodium salt of DNA with a molecular weight of 270-500 × 10 ³ Yes at weight ratios of nucleotides, in mass%: adenine - 29, thymine - 27, guanine - 22, cytosine - 20. The method does not cause complications and at the same time stimulates bone marrow hematopoiesis [19].

This method also has relatively low treatment efficacy.

There is also a known method of leukostimulation in the treatment of generalized tumors by intramuscular or subcutaneous injection of a pre-prepared mixture of a cytostatic and a leukostimulator, with 5-fluorouracil being used as a cytostatic and a sodium salt of DNA as a leukostimulator. As the sodium salt of DNA, Derinat can be used. The method improves the effectiveness of treatment by changing the transportation of 5-fluorouracil, maintaining the concentration of cytostatic in the tumor tissue [20].

This method also has relatively low treatment efficacy.

A method is also proposed for using a three-component conjugate consisting of the Derinat leukostimulator, an antibiotic, and blood proteins, which makes it possible to deposit anthracycline antibiotics intramuscularly or subcutaneously in an inactive form. The absence of cardiotoxicity and hematotoxicity allows the use of this method at the stage of generalization, in conditions of persistent leukopenia or severe general condition of patients [21].

This method has a relatively low treatment efficiency. The method of using exogenous DNA preparation for the treatment of patients with advanced forms of tumors of the abdominal organs is described by intramuscular injection of a mixture of native DNA and an anthracycline antibiotic containing 5 mg of anthracycline antibiotic dissolved in 4 ml of solvent, 75 mg of DNA and 2 ml of 0.5% novocaine solution (total for a course of 50 mg of an anthracycline antibiotic) and activation of the antitumor cells of the host organism (CD 16 natural killer cells) by extracorporeal connection of a donor pork spleen. The method allows to enhance the effect of the treatment of patients with advanced forms of tumors of the abdominal cavity [22].

This method has a relatively low treatment efficiency.

Also described is a method of using an exogenous DNA preparation in the treatment of malignant neoplasms, comprising administering the DNA intramuscularly, from 1 to 3 times a day, with a total amount of 0.1-0.5 g, and in taking anti-tumor phytopreparations. Additionally, antitoxic, and / or antianemic, and / or tonic, and / or antihistamine, and / or immunomodulating and / or diuretic phytopreparations are administered. Treatment with this method has a differentiated effect on normal and pathologically altered cells, as a result of which stimulation of the proliferation and differentiation of normal cells occurs along with the induction of apoptosis mechanisms in transformed cells [23].

This method has a relatively low treatment efficiency.

There is another method of using the exogenous DNA preparation in the treatment of malignant neoplasms, which, depending on the nature and severity of the disease, the patient is injected with parenteral sodium salt of DNA, in a single dose of 0.5-4.0 mg / kg once every four days , in combination with the introduction of human alpha-fetoprotein, in a single dose of 500-5000 IU / kg 1-2 times a day daily, for a course of 14-30 days. Alpha-fetoprotein is administered subcutaneously, intramuscularly or intravenously, and DNA is administered intramuscularly [24].

This method has relatively low treatment efficiency, these patented methods also have limited applicability, since they focus either on leukostimulation using injection forms of drugs of foreign origin, in which the DNA molecule is hydrolyzed to low molecular fractions and, in some cases, denatured with subsequent uncontrolled reassociation, which violates the native structure of the active principle of the drug, or are used in combination with other anti-cancer drugs eparatami without any logical explanation for the mechanism of action.

In addition, none of the above methods of using DNA preparations activates the anti-cancer immunity of the body.

Currently, an active search continues for new drugs that stimulate leukopoiesis and drugs that induce anti-cancer adaptive immunity.

Known methods of using the preparation of exogenous human DNA "Panagen", which has both antitumor, antitoxic and radioprotective effects. The Panagen preparation contains a native, species-specific fragmented nucleoprotein complex, consisting of double-stranded DNA and associated nuclear matrix proteins, which allows to expand the spectrum of therapeutic activity of the drug [25].

This method using fragmented human DNA Panagen has limited applicability.

A method for the use of the Panagen human DNA preparation in the treatment of cancer by using homologous DNA having a biologically active size, comprising the complete genome of a physiologically and genetically healthy donor, is described. DNA is administered to a patient in a dose that creates such a concentration of DNA in blood plasma that is equal to or exceeds the concentration of the blood plasma DNA itself, but does not exceed the maximum allowable concentration of 30 μg / ml. EFFECT: invention provides natural homologous DNA repair with replacement of chromosome regions carrying mutations that lead to cell okraization without undesirable effects when using DNA at the indicated dose [26].

This method using fragmented human DNA Panagen also has limited applicability.

A known method of using the preparation of human DNA in the treatment of hematopoiesis disorders associated with chromosome defects and subsequent death of SSC during therapy with cytostatics or high doses of radiation, treatment of diseases associated with impaired mechanisms of SSC differentiation. The method is based on introducing fragmented DNA, which is allogeneic, into the patient’s body, obtained from genetically and physiologically healthy donors and consists of fragments whose length corresponds to 1-10 nucleosome units making up the patient’s complete genome, in association with nuclear matrix proteins. Enter such a quantity of fragmented DNA in order to achieve a concentration of fragmented DNA in the patient’s blood plasma equal to or higher than the concentration of the blood plasma’s own DNA, but not more than 1500 ng / ml. The method allows to restore violations of the CCM DNA of a patient by replacing damaged sections of chromosomes with homologous recombination and provides an extension of the scope of the fragmented DNA preparation [27].

The method also has limited applicability.

A number of publications describe a possible mechanism for the leukostimulating effect of Panagen. For example, the source [28] disclosed that DNA fragments delivered into the intranuclear space of CD34 + CCMs activate a pool of hierarchical kinases with their double-stranded ends, while the molecular cycle cell arrest machine is launched in CCMs resting at this time. After some time, the repair system removes these fragments from the nuclear space, but the cell has already entered the activated state and must advance along the cell cycle with its subsequent completion. With the constant delivery of DNA fragments into the nuclear space of this type of cells, the division of these cells will be continuously activated, which determines the leukostimulating effect of exogenous DNA.

Revealed publications suggesting that injections of exogenous fragmented genomic DNA are associated with effects that affect the functional integrity of the CCM. Thus, in the experiments performed in [29], the appearance of splenic colonies in deadly irradiated mice after timely injection of exogenous DNA was noted. This observation indirectly indicated that exogenous DNA, upon intraperitoneal administration, reaches bone marrow cells and acts on CD34 + CCMs, while maintaining their viability. Descendants of the surviving blood stem cells form colonies in the spleen that revive the immune system of the experimental animal.

All these methods of using the preparation of fragmented human DNA Panagen have limited applicability, since they do not take into account in one strategy the entire combination of the discovered therapeutic properties of the drug.

The closest in essence to the proposed one is a method of treating oncological diseases, based on the introduction into the patient’s body of a preparation of the cytostatic group of cyclophosphamide, which is administered in doses of 20 to 100 mg / kg of the patient’s weight, and the preparation of fragmented allogeneic double-stranded genomic DNA with fragments having biologically active size and constituents of the full genome of a physiologically and genetically healthy donor (exogenous DNA), while the exogenous DNA preparation is administered 48 hours after each injection of the drug of the cytostatic group and for the next 30 days for the regimen when the patient received a full course dose of 6-14 g, or for the next 3-5 days after a single injection and a day before the next injection, continuing such an intermittent regimen until complete therapeutic or maintenance doses of cytostatic [30].

To implement the known method, the Panagen preparation can be used (LSR No. 004429/08 dated 06/09/2008), which is used as a substrate for activation of maturation, induction of antigen-presenting and specific allostimulatory activity in the Th1 direction of the antigen-presenting DC of the body. In this case, a dosage form of the drug in the form of tablets containing 5 mg of the active substance can be used, for example, according to the scheme 30 minutes and 2, 3 and 5 days after the administration of cyclophosphamide in the amount of one tablet in the first 30 minutes after the administration of cyclophosphamide and then in the indicated days, one tablet every 2-3 hours during the day (wake time), but not more than the maximum allowable amount equal to 30 mg of the active substance per day.

However, the closest method has a relatively low treatment efficiency, due to the fact that only one direction of exposure of exogenous DNA to the patient’s body is used and the effect of other effective therapeutic properties, which are manifested in combination with the action of the cytostatics of cyclophosphamide and doxorubicin, is not provided.

The desired result is to increase the effectiveness of treatment.

The desired result is achieved in that in a method based on the use of cytostatic group preparations in stage II-IV breast cancer treatment regimens, additionally after cyclophosphamide (500 mg / m ² ) and doxorubicin (50 mg) once in a single cycle of chemotherapy / m ² ) (AC scheme) or cyclophosphamide (500 mg / m ² ), doxorubicin (50 mg / m ² ) and fluorouracil (500 mg / m) (FAC scheme), the patient is prescribed a drug based on exogenous human Panagen DNA (LSR No. 004429/08 dated 06/09/2008) in the form of tablets coated with a gastroenteric covered, at a dose of 30 mg / day with a uniform dose during the active period of the day, which is one tablet of the Panagen preparation six times a day (every two hours) with the start of the drug immediately after chemotherapy in the amount of 3 tablets for 6 hours , a break in taking the drug, resuming it after 42 hours, that is, 48 hours after chemotherapy (day 3), continuing to take the drug for 17 days to 20 days after chemotherapy (inclusive), and continuing to take rata according to the scheme until all chemotherapy.

In addition, the desired result is achieved by the fact that the use of drugs of the cytostatic group when they are synergistically combined with Panagen according to this scheme is a new strategy for the treatment of breast cancer, the use of which leads to the simultaneous manifestation of three types of therapeutic effects within the same chemotherapy course: preservation and stimulation of granulocyte-macrophage and lymphocytic hematopoietic sprouts, which compensates for the negative leukoreducing effect of cytostatics, to preservation of innate anti-cancer immunity at the level prior to cytostatic therapy and the induction of anti-cancer adaptive immunity characterized by an increase in the peripheral blood of patients with cytotoxic CD8 + lymphocytes with a high content of perforins.

More broadly, the objective of the present invention is to develop a justified and experimentally confirmed method (strategy) for simultaneous exposure of breast cancer using multiple courses of programmed chemotherapy to tumor tissue, reducing its development by activating DC and inducing the development of adaptive immunity, and on the patient’s body, helping him survive the negative effects of powerful cytostatics, which are cyclophosphamide and doxorubicin account patronage and stimulation of proliferation of hematopoietic precursor bone marrow of varying degrees of ripeness.

The following are theoretical and experimental data confirming that the invention meets the criteria of novelty, inventive step and practical (industrial) applicability.

The proposed method for the treatment of cancer is implemented as follows.

A method of treating cancer is based on the use of an exogenous Panagen DNA preparation in the form of tablets coated with a gastroenteric coating in treatment regimens for stage II-IV breast cancer using a cyclophosphamide (500 mg / m ² ), doxorubicin, which is a single chemotherapy cycle. (50 mg / m ² ) and fluorouracil (500 mg / m ² ). The drug Panagen is used at a dose of 30 mg / day with a uniform fractional intake during the active period of the day, which is one tablet of the Panagen drug six times a day (every two hours) with the start of the drug immediately after chemotherapy in the amount of 3 tablets for 6 hours, a break in taking the drug, resuming it after 42 hours, that is, 48 hours after chemotherapy (day 3) and continuing for 17 days to 20 days after chemotherapy (inclusive). With this effect, leukostimulation and activation of adaptive immunity occur simultaneously in the patient's body.

To confirm that the proposed method allows to achieve the desired result, we present its theoretical and experimental justification. The proposed method (strategy) for the treatment of breast cancer is based on two effects associated with the simultaneous effect of fragmented exogenous DNA (the active principle of the Panagen drug) on SSCs and immunocompetent DCs, which simultaneously leads to the induction of proliferation of the former and activation of the professional properties of the latter. Consider the theoretical and experimental evidence of both properties of the Panagen preparation.

The drawing shows:

figure 1 - Postembryonic hematopoiesis (scheme for NA. Yurina). Stages of blood differentiation: I-IV - morphologically unidentifiable cells; V, VI - morphologically identifiable cells; B - basophil; PFU - burst forming unit; G - granulocytes; Gn - neutrophilic granulocyte; CFU - colony forming units; CFU-S - splenic colony forming unit; L - lymphocyte; Lsk - lymphoid stem cell; M is a monocyte; Meg - megakaryocyte; Eo - eosinophil; E - erythrocyte [31].

figure 2 - Delivery of exogenous DNA in CMC in vivo. A - Electrophoregram (left) and X-ray (right) of the genomic DNA of KKM mice after i.v. administration of labeled human Alu repeat following injection of cyclophosphamide (CF) (1) and as a single drug (2) labeled with HpaI salmon repeat following injection of CF (3) both in the form of a single drug (4), labeled plasmid pEGFP-N1 against the background of CF injection (5) and as a single drug (6), and also after the intravenous administration of labeled dATP against CF injection (7) and as a single drug (8). M is a marker of molecular weight λHindIII. B - PCR on a slide with biotin-labeled nucleotides and using human DNA, fragmented to sizes of 20-500 bp, as a primer. KKM nuclei of intact mice and after a 12-fold (18 hours after the administration of CF) hourly iv administration of 0.5 mg of human DNA. B - Electrophoregram (left) and x-ray (right) of PCR products with primers for a unique human caspase 3 gene using human DNA (1), bone marrow cell DNA from CBA mice (2) and mouse bone marrow fraction DNA from a mouse, isolated 8 hours after the introduction of human DNA according to the scheme described above (3). A fragment of ~ 260 bp, labeled in PCR with primers for the human caspase 3 gene and a human DNA template, was used as a probe for hybridization. 2% agarose gel. M is a molecular weight marker (100 bp). The arrow denotes a hybridizing fragment corresponding to a PCR product with primers for the human caspase 3 gene. D - FISH with biotin-labeled Alu repetition of human mouse CMC preparations prepared 38 hours after CF injection (200 mg / kg), against the background of the introduction of human DNA according to the previously mentioned scheme and without it, as well as human fibroblasts. Arrows indicate hybridization zones.

figure 3 - Delivery of exogenous DNA in CMC in vitro. A - Results of incubation of CMC with Alu DNA fragment with a 300 bp repeat synthesized and labeled in PCR, in an amount of 0.5 to 32.5 μg. Electrophoregram (left) and x-ray (right) after pouring in blocks of low-melting agarose and electrophoresis of KKM nuclei of the mouse isolated 18 hours after exposure to CF and intact mouse. M is a marker of molecular weight λHindIII. On the right is a quantitative assessment of the labeled material contained in the KKM nuclei. The assessment was carried out using the Molecular Imager FX Pro + phospho-image manager and the Quantity One program. B - The results of incubation of CMC with plasmid pEGFP-N1, hydrolyzed with restriction enzyme HindIII and labeled at the sticky end, for various times - 1, 2, 4 and 7 hours. Electrophoregram (left) and x-ray (right) after pouring in blocks of low-melting agarose and electrophoresis of KKM nuclei of the mouse isolated 18 hours after exposure to CF and intact mouse. M - molecular weight markers. On the right is a quantitative assessment of the labeled material contained in the KKM nuclei. The assessment was carried out using the Molecular Imager FX Pro + phospho-image manager and the Quantity One program. C - Results of incubation of CMC with DNA of plasmid pEGFP-N1, hydrolyzed with restriction enzyme Smal (1), restriction enzyme HindIII (2), supercoiled form of plasmid pEGFP-N1 (3) and in the absence of exogenous DNA (4) for 7 hours. Electrophoregram (left) and X-ray (right) after pouring in blocks of low-melting agarose and electrophoresis of mouse CMC nuclei isolated 18 hours after exposure to CF and an intact CBA mouse. P1 and P2 are supercoiled and linearly shaped pEGFP-N1 plasmid DNA, and M is a molecular weight marker.

figure 4 - Internalization of exogenous DNA in CD34 + CCM. CMC preparation isolated 18 h after injection to CF mice (200 mg / kg) and cultured ex vivo with Alu DNA of a human repeat labeled with TAMRA fluorochrome. Additionally, cells were stained with specific FITC-labeled antibodies to the surface marker CD34. DAPI — staining of nuclear chromatin, FITC — staining with specific antibodies to the surface marker of CD34 + pluripotent cells, TAMRA — detection of exogenous DNA labeled with fluorochrome TAMRA in PCR nuclei.

figure 5 - Stimulation of leuko and erythropoiesis. A - Male mice of CBA line for 6 days daily were injected intraperitoneally with 50 μg of DNA of various origin isolated from salmon milk, human placenta and organs of CBA mouse mice, in 0.2 ml of nat. solution. CF at a dose of 200 mg / kg of body weight was administered once into the abdominal cavity on the second day after the first DNA injection. The number of leukocytes in the blood was individually counted in each mouse at 3, 5 and 7 days after DNA injection. Blood was taken from the tip of the tail. It can be seen that on the 3rd day after CF injection, the number of leukocytes in the blood was 5-6% of the original. On day 5, in the group of mice receiving one CF, the number of leukocytes did not reach the initial level, while in mice receiving DNA, their recovery to normal was observed. On day 7, when the leukocyte level in the groups of mice that received human and mouse DNA was more than 1.5-2 times higher than the initial level, in the group that received salmon DNA, their level did not change compared to the previous measurement. Thus, exogenous homologous DNA stimulates the restoration of the number of leukocytes in the blood of CBA mice after a single injection of CF, which inhibits hematopoiesis. B - Human bone marrow mononuclear cells (MNCs) were incubated for 1 hour at 37 ° C with human exogenous DNA at a concentration of 100 μg / mg. In the control, cell culture medium was added instead of DNA in the appropriate amount. On the 8th and 14th day after incubation, the number of erythroid colonies formed in the cell culture was estimated. After preincubation of MNCs with exogenous DNA, a significant, statistically significant increase in the number of generated erythroid colonies is recorded (p <0.001). The most demonstrative increase in the number of erythroid colonies after processing of exogenous human DNA is observed in the early stages - on the 8th day of cultivation, when the number of erythroid colonies formed in the experiment exceeds the number of those in the control by 3-4 times or more. When using the model of erythroid colony formation, the effect of the preparation of human exogenous DNA on damaged hematopoietic precursors in 40% of cases is not quantitative, but qualitative. At the same time, exogenous DNA was the active principle that allowed the predecessors to maintain (restore) viability. The mechanism that explains the rescue of precursors as a result of exposure to exogenous DNA fragments is not known.

figure 6 - Dynamics of tumor growth Krebs-2 in mice after an XT.

Fig.7 - Percentage of CD8 + perforin + T cells in intact MNCs and after incubation with DC matured in the presence of LPS, DNA or in the absence of a maturing stimulus (0).

on Fig - Control points of the study. * - Blood test for the absolute number of leukocytes and neutrophils, a general blood test. # - Counting the number of CD34 + cells.

figure 9 - the Absolute number of leukocytes at the starting point 0 and determined at control points 7, 14 and 21 days after XT. There was a significant decrease in the number of cells in the peripheral blood of patients participating in the tests at the control point for 14 days relative to the initial level. ** - differences from the placebo group are significant with a probability p _u <0.01.

figure 10 - Conventional leukocyte count relative to the corresponding control point after 1 XT in the groups "Placebo", "Panagen-responding" and "Panagen-not responding" on 21 days after the XT. ** - differences from the placebo group are significant with a probability p _u <0.01.

figure 11 - Comparison of the conditional values of the content of leukocytes in each group in the second year of XT at control points 14 and 21 days after the introduction of cytostatic relative to the parameter value after 1 XT. The Panagen-responding and Panagen-non-responding groups were formed independently for each control point.

on Fig - Comparison of the conditional values of the content of leukocytes in each group in the third year of XT at control points 14 and 21 days after the introduction of cytostatic relative to the parameter value after 1 XT. The Panagen-responding and Panagen-non-responding groups were formed independently for each control point. ** - differences from the placebo group are significant with a probability p _u <0.01.

on Fig - Comparison of the conditional values of the content of neutrophils on the 14th day after the introduction of cytostatics (control point) after 2 and 3 XT relative to the parameter value after 1 XT.

on Fig - Comparison of the conditional values of the content of neutrophils on day 21 after the introduction of cytostatics (control point) after 2 and 3 XT relative to the parameter value after 1 XT.

on Fig - Comparison of the conditional values of the content of neutrophils in each group in the course of courses XT at the control point 14 days after the introduction of cytostatic in relation to the indicator 1 XT.

in Fig.16 - Comparison of the conditional values of the content of neutrophils in each group along the course of XT courses at the control point 21 days after the introduction of cytostatic in relation to indicator 1 XT.

on Fig - Comparison of the conditional values of the content of neutrophils in each group along the course of XT courses at control points 14 and 21 days after the introduction of cytostatic in relation to the original value.

in Fig.18 - Comparison of the conditional values of the neutrophil content in each group in the second year of XT at control points 14 and 21 days after the introduction of cytostatic relative to the parameter value after 1 XT. The Panagen-responding and Panagen-non-responding groups were formed independently for each control point.

on Fig - Comparison of the conditional values of the content of neutrophils in each group in the third year of XT at control points 14 and 21 days after the introduction of cytostatic relative to the parameter value after 1 XT. The Panagen-responding and Panagen-non-responding groups were formed independently for each control point. ** - differences from the placebo group are significant with a probability p _u <0.01.

on Fig - the frequency of manifestation of neutropenia of all degrees in patients at various control points during 3 courses of XT.

on Fig - Frequency of manifestation of neutropenia of the II degree in patients at various control points during 3 courses of XT.

on Fig - Dynamics of the recovery of neutrophils after the largest drop in their number in the peripheral blood to values that induce febrile neutropenia, in the placebo and Panagen groups without additional leukostimulation.

in Fig.23 - The absolute number of monocytes at the starting point 0 and determined at control points 7, 14 and 20 day after the XT. A significant decrease in the number of cells in the peripheral blood of patients participating in the tests at the control point of 14 days is noted.

on Fig - Relative number of monocytes in groups at control points in comparison after 2 and 3 XT.

on Fig - Comparison of the relative number of monocytes within each group at control points along the course of XT courses. It can be noted that in the Panagen-responding group, the relative content of monocytes is constantly growing from XT to XT. In the placebo group, the increase in the number of monocytes in peripheral blood to 3 XT stops. The results obtained indicate the growing role of the drug in the preservation and activation of proliferation of precursors of the hematopoietic monocytic sprout.

on Fig - Comparison of the conditional values of the content of monocytes in each group in the second year of XT at control points 14 and 21 days after the introduction of cytostatic relative to the parameter value after 1 XT. The Panagen-responding and Panagen-non-responding groups were formed independently for each control point. ** - differences from the placebo group are significant with a probability p _u <0.01.

on Fig - Comparison of the conditional values of the content of monocytes in each group in the third year of XT at control points 14 and 21 days after the introduction of cytostatic relative to the parameter value after 1 XT. The Panagen-responding and Panagen-non-responding groups were formed independently for each control point. * - differences from the placebo group are significant with a probability p _u <0.05.

on Fig - Comparison of the conditional content of lymphocytes in the peripheral blood of patients participating in the study at the last control point 21 days of the third XT. The significant difference between the Placebo and Panagen groups is noteworthy. The data obtained indicate the preservation and maintenance of the level of lymphocytes during XT, which reflects the preservation of the immunocompetence of the body of patients taking the drug Panagen. * - differences from the placebo group are significant with a probability p _u <0.05.

on Fig - Relative number of lymphocytes in groups at control points in comparison with the level after 1 XT, which was taken as 100%, after 2 and 3 XT. Significantly, at the control point for 21 days, the number of lymphocytes in the Panagen-responding group was greater than in the Placebo group. At all control points of the second and third XT, there is a higher number of lymphocytes in the group of “responding” patients. * - differences from the placebo group are significant with a probability p _u <0.05; ** - differences from the placebo group are significant with a probability p _u <0.01.

on Fig - Comparison of the relative number of lymphocytes within each group at control points along the course of XT courses.

on Fig - the relative number of CD34 + / 45 + cells in the peripheral blood of patients on 21 control days after 3 courses of XT in relation to the initial level.

on Fig - the relative number of CD34 + / 45 + cells in the peripheral blood of patients on the 7th control day after the first XT in relation to the initial level. ** - differences from the placebo group are significant with a probability p _u <0.01.

on Fig - Comparison of cytotoxicity indices (reflects the state of the natural killer system in the body) in patients in the study. With reliability p _u <0.01, natural killers maintain their specific functions at the level before therapy in patients taking Panagen after three courses of XT. In patients of the placebo group, the activity of natural killers by the end of 3 XT drops to 30% of the original.

on Fig - Comparison of the conditional values of the content of different cells in each group on 14 and 21 days after 3 XT relative to the parameter values after 1 XT. * - differences from the placebo group are significant with a probability p _u <0.05; ** - differences from the placebo group are significant with a probability p _u <0.01.

on Fig - Conditional content (%) of CD11-CD123 + plasmacytoid DC in the peripheral blood of patients in the study, 21 days after 1 and 3 XT relative to the initial level.

on Fig - Conditional content (%) of CD11-CD123 + plasmacytoid DC in the peripheral blood of patients in the study, 21 days after 3 XT relative to the level after 1 XT.

on Fig - Conditional content (%) of CD11 + CD123-myeloid DC in the peripheral blood of patients in the study, 21 days after 1 and 3 XT relative to the initial level.

in Fig.38 - Conditional content (%) of CD11 + CD123-myeloid DC in the peripheral blood of patients in the study, 21 days after 3 XT relative to the level after 1 XT.

in Fig.39 - Conditional content (%) of CD8 + perforin + cytotoxic lymphocytes in the peripheral blood of patients in the study, 21 days after 1 and 3 XT relative to the initial level. * - differences from the placebo group are significant with a probability p _u <0.05.

on Fig - Conditional content (%) of CD4 + CD25 + Fox3 + T-regulatory lymphocytes in the peripheral blood of patients in the study, 21 days after 1 and 3 XT relative to the initial level. ** - differences from the placebo group and the group of patients in whom the number of T-regulatory lymphocytes has not changed, are significant with a probability p _u <0.01.

The proposed method for the treatment of cancer is implemented as follows.

First, we provide information confirming the effectiveness of the proposed method for the treatment of cancer.

In oncological practice, the use of cytostatic drugs is one of the main methods of treating tumors of various origins. The mechanism of action of all cytostatics is based on blocking the vital biochemical metabolic pathways of the cancer cell, which ultimately leads to its death. Along with cancer cells, all actively proliferating cells of the body, and primarily hematopoietic cells, are exposed to the action of the cytostatic agent. Inhibition of the activity of bone marrow precursors of hematopoiesis leads to the development of leukopenia and neutropenia of various gravity. This toxic effect of the action of cytostatics in the structure of XT complications occupies one of the leading positions. Neutropenia of the II-IV degree is the main limiting factor preventing the onset of XT, causing the need to reduce the doses of chemotherapy drugs, delay and / or cancel treatment courses (table 1).

Table 1 The degree of leuko- and neutropenia in accordance with the WHO classification. Leukopenia The number of leukocytes in 1 μl of blood Degree of neutropenia The number of neutrophils in 1 μl of blood I 3900-3000 I 2000-1500 P 2900-2000 II 1500-1000 III 1900-1000 III 1000-500 IV <1000 IV <500

Neutropenia is associated with a sharp decrease in the number of neutrophils circulating in the peripheral blood, and most often develops on the background or shortly after intensive regimes of cytostatic XT for various malignant neoplasms. An extreme degree of neutropenia - febrile neutropenia - can be a hyperergic reaction of the body to the toxic effects of cytostatic chemotherapy drugs, the products of the destruction of tumor and healthy cells, or a sharp decrease in the number of circulating neutrophils and impaired production of cytokines and immunoglobulins. However, most often, febrile neutropenia is a manifestation of an infection that is activated as a result of a decrease in control of the immune system caused by a critical decrease in the number of immunocompetent cells in peripheral blood. The infectious process induced by neutropenia is extremely difficult. Infection quickly spreads from the primary focus to other organs and systems and quickly leads to the death of the patient even in cases where the infectious microorganism is saprophytic or low-virulent for patients with normal immunity and the normal number of neutrophils circulating in the blood. In this regard, the activation of a population of hematopoietic stem cells (HSCs) and the induction of their proliferation (in particular, the induction of leukopoiesis and neutropoiesis) is one of the main methods of combating leuko- and neutropenia caused by intensive regimes of cytostatic treatment in cancer treatment regimens.

In modern medicine, there are several groups of drugs that stimulate leukopoiesis. These are medicines

- stimulating metabolic processes - methyluracil, pentoxyl, leucogen, dicarbamine and others used to treat various forms of leukopenia,

- and the so-called colony-stimulating factors - filgrastim, sagramostim, lenograstim, etc.

Pyramidine derivatives - methyluracil (syn. Metacil) and pentoxyl - have the ability to stimulate erythro- and especially leukopoiesis, as well as cellular and humoral immunity. In addition, anabolic activity is also expressed in the preparations, therefore, they accelerate the processes of cell regeneration and contribute to faster healing of wounds. In clinical practice, methyluracil and pentoxyl are used to stimulate leukopoiesis in case of chronic poisoning with benzene, alimentary toxic aleukia (food poisoning caused by eating grains infected with Fusarium sporotrichiella fungi), agranulocytic angina (characterized by peripheral granuloma syndrome) leukopenia developed as a result of x-ray or radiotherapy. In some cases, a leukogen derivative is used to treat these leukopenias, which is used for the same indications as pyramidine derivatives.

The hematoprotective effect of dicarbamine in myelosuppressive XT is due to accelerated maturation of neutrophilic granulocyte precursors. The drug accelerates the formation of detoxifying granules in the progenitor cells of the myeloid germ, responsible for the state of anti-infection immunity. Along with this, dicarbamine reduces the intensity of programmed cell death (apoptosis) by a factor of 3-4 in the myeloid precursor population. This leads to a significant increase in the number of karyocytes and the redistribution of cells towards functionally complete forms. It causes a decrease in the incidence of limiting leukemia and neutropenia. Allows to carry out treatment as scheduled without reducing the doses of cytostatics and the risk of hematological complications.

For the treatment of severe forms of leukopenia, drugs obtained by genetic engineering are widely used, which are an analogue of the human granulocyte colony stimulating factor, such as mgramramostim (syn. Leukomax), lenograstim, filgrastim, etc. These drugs are structural and functional analogues of granulocyte colony stimulating factor responsible for the process of mobilization of HSCs, and regulate the production and release of neutrophils into peripheral blood. A granulocyte colony-stimulating factor is used to treat leukopenia, mainly various types of neutropenia in patients receiving myelosuppressive XT for oncological diseases, to treat myelodysplastic syndrome, improve tolerability of immunosuppressive drugs in bone marrow transplantation, and improve the immune status of patients suffering from AIDS and other infections.

Recently, drugs with the active substance of nucleic acids (ridostin, derinat, polydane, deoxinate, etc.) are finding wider application in the practice of treating cancer and viral diseases. One of the directions of the effect of these drugs on the body is the leukostimulating activity they show. The mechanism of the leukostimulating effect of these drugs is unknown. Derinat is one of the representatives of DNA-based drugs. Indirect hemopoiesis stimulator. It causes an increase in the production of endogenous colony-stimulating factors. Helps increase platelet count. Reduces the degree of immunosuppression after chemotherapy and radiation therapy, increasing the content of CD4 +, increases their share in the ratio of immunoregulatory subpopulations (CD4 + / CD8 +), while maintaining a high proliferative activity of T-lymphocytes. Significantly reduces the level of superoxide dismutase and malondialdehyde, showing moderate antioxidant activity. Accelerates the differentiation and functional maturation of neutrophils.

The use of cytostatics acts destructively both on committed precursors of leukopoiesis, and primarily on the first echelon of CD34 + CCM progenitors. First of all, the possibility of a quick replenishment of the blood cells of the lymphoid row after an XT depends on the state of this population of cells. In this regard, the main issue of post-chemotherapeutic recovery is the development of approaches and the creation of new drugs aimed

- the induction of proliferation of CCM in the niches of the bone marrow;

- the mobilization of CD34 + CCM and the associated proliferation of committed precursors of neutropoiesis and the release of cells into the peripheral bloodstream.

And, thus, there are two ways to increase the number and activity of neutrophils in the body. The first is to induce the mobilization of G-CSF, which simultaneously leads to the activation of proliferation of committed neutrophil precursors and the entry of CD34 + HSC into the bloodstream, and the second is to induce HSC proliferation directly in hematopoietic niches (in situ). Both of these molecular processes are called leukostimulation.

Figure 1 shows the modern scheme of hematopoiesis. The polypotent SCC-HSC (blood stem cell, hematopoietic stem cell) has such basic characteristics as the ability to long-term self-maintenance, high proliferative potential, and the ability to differentiate different lines of myelo- and lymphopoiesis into cellular elements. The content of CCM in the human bone marrow in the postnatal period is several tenths of a percent of the total number of myelocaryocytes. Normally, the bulk of the CCM (95%) is in the phase of the G0 cell cycle. Studies in mice have shown that pluripotent bone marrow SSCs are divided into two types: more primitive cells, capable of self-reproduction over a long period of time and providing long-term, albeit slow, recovery of hematopoiesis, and more mature cells, providing quick, but only short-term restoration of blood formation.

According to the generally accepted hematopoietic scheme, the lymphoid stem cell gives rise to the development of B- and T-lymphocytes, and, probably, natural killer cells. From a precursor cell of myelopoiesis of a colony forming unit of granulocytes, erythrocytes, monocytes, megakaryocytes, myelomonocytic progenitor cells arise, which, in turn, are responsible for the production of granulocytes, monocytes / macrophages, megakaryocytic and erythroblastic progenitor cells. It has been established that DCs arise both from a common myelopoiesis precursor cell and from a common lymphopoiesis precursor cell. It is assumed that the differentiation of most types of stem cells occurs according to the principle of phased hierarchical maturation through intermediate intensively proliferating progenitor cells. The precursor polypotent cells and the unipotent cells formed from them form colonies from cells of only one line. A polypotent progenitor cell has significant ability to reproduce. Being stimulated by growth factors, it is able to make up to 13 mitoses and form several tens of unipotent cells, from which several thousand committed descendants are formed. So, for example, from each colony-forming unit of the erythroid row, up to 50 red blood cells are formed. The colony-forming unit of the granulocyte-macrophage series is capable of 5-6 divisions, the unipotent precursors of granulocytes and macrophages formed from it - another 5-6 divisions each. This allows one granulocyte-macrophage polypotent cell to form thousands of mature progeny cells - granulocytes and monocytes. Even one CCM (HSC) can provide long-term, throughout life, maintenance of the production of hematopoietic cells. Apparently, among HSCs, there are cells with infinite proliferative potential, the ability to unlimited reproduction and production of an arbitrarily large number of blood cells. This is a very rare category of cells that occurs in the bone marrow at a concentration of about 1 per 100,000 cells.

Studies of the hematopoiesis kinetics of mice show that hematopoiesis is supported mainly by a small number (several tens) of relatively short-lived, successive cell clones. The life span of an individual clone in the bulk is small and usually does not exceed 1 month. As a rule, the composition of the hematopoietic tissue completely changes within 1-4 months, and only very rare clones, about 10% of them, exist for more than 3 months. The disappeared clones never appeared again, which indicates the depletion in the process of hematopoiesis of the original stem cell for a given clone. As a rule, the size of an individual clone is usually less than 0.5-1 million mature cells.

Thus, the multi-stage process of hematopoiesis, which ends with the emergence of mature cellular forms (red blood cells, leukocytes, platelets) into the circulating blood, is supported by hematopoietic stem cells or hematopoietic cells (CCM, HSC) embedded in ontogenesis. As a result of the proliferation of HSCs leaving the resting state and their subsequent differentiation, the direction of which is chosen stochastically, daughter clones of committed progenitor cells alternate, and then morphologically recognizable cells of various sprouts of hemo- and lymphopoiesis are formed.

The leukostimulating effect of the drug based on human Panagen DNA is based both on direct stimulation of cell division (by the mechanism of activation of CCM by double-stranded ends of exogenous DNA introduced into the nucleus [32], and indirectly, through the induction of specific cytokines. As part of the scientific and experimental substantiation of the clinical trials, large-scale long-term studies have been conducted aimed at understanding the mechanism of HSC activation and the induction of their proliferative activity. ah, indicate that fragments of exogenous DNA reach the nuclear space of CMC, including CD34 + HSC, in the form of exogenous polymeric material (Fig.2-4) [33].

The experiments on activation of human CD34 / 45 + HSC in ex vivo system showed the following results. Hematopoietic precursors were planted on a medium containing conventional growth factors, including colony-stimulating factors, as well as fragmented human DNA (Panagen preparation). The presence of exogenous DNA in methylcellulose led not only to the induction of aggressive proliferation of stem cells, but primarily their “revival” (Fig. 5).

The proposed mechanism of proliferation induction is associated with the appearance of double-stranded DNA fragments in CD34 + SSC. It is known that the presence of more than 60 double-stranded ends in the nuclear space of a cell leads to activation of the cell’s observing system (ATM, ATR, DNA protein kinase) [32]. A cell that is still at rest (GO) is activated in the direction of arrest of the cell cycle, after which synthesis and mitosis follow. If enough exogenous DNA fragments are continuously present in the bloodstream, they will constantly “irritate” the SSC, induce movement along the cell cycle, which will lead to its continuous division. As mentioned above, in the bone marrow, 95% of HSCs are in GO state. If the proposed concept is correct, then this means that when the bone marrow space is saturated with exogenous DNA fragments, the entire population of pluripotent precursors will have to go through or repeatedly go through the cell cycle. Such a proliferative impulse can multiply the number of committed descendants of all types of hematopoietic sprouts. In our opinion, this is one of their possibilities of activating the proliferation of CCMs by double-stranded ends of nucleic acid fragments. Regarding the mechanism of “revitalization” of HSCs after cryopreservation, we believe that this effect is associated with activation of recombination processes in thawed HSCs against the background of the presence of exogenous DNA as a matrix for reparative recombination [32].

Studies have been conducted to analyze the effect of exogenous DNA on the activation of antigen-presenting properties of DCs. It was shown that fragments of exogenous DNA induce maturation and allostimulatory activity of DCs in the Th1 direction [34–37]. A pool of cytotoxic perforin-containing CD8 + T-lymphocytes is formed.

To assess the effectiveness of the effect, Krebs 2 tumor was used, which reached 1 cm ³ in size (Fig. 6). CBA mice were intramuscularly inoculated with a Krebs-2 tumor (ascites 1:10 dilution). On the 8th day of tumor growth, 5 groups of animals were formed and preparations were introduced: 1 - control; 2 (n = 6) - mice were injected with CF in a dose of 100 mg / kg; 3 (n = 7) - iv doxorubicin (DR) 3 mg / kg and ip CF 100 mg / kg, every other day ip. solution for 3 days; 4 (n = 7) - in / in DR 3 mg / kg and in / b CF 100 mg / kg, every other day in / b DNA of 500 μg in 200 μl phys. solution for 3 days; 5 (n = 2) - in / in DR at 3 mg / kg.

On day 17, 2 mice were scored from each group; inguinal lymph nodes, thymuses, and spleens were taken from them. Cells were carefully gutted from the organs, resuspended and counted, spleen cells were further fractionated on a density gradient of ficoll-urographin to isolate the mononuclear fraction of blood. Cells of different organs in each group of mice were mixed in equal amounts. The obtained cell fractions were tested by antibody staining for surface markers of DC maturity and cytotoxic lymphocyte populations.

Analysis of DC surface markers was performed in a population of cells that did not carry a surface marker of progenitor cells (CD34-), i.e. the analyzed cell population was differentiated (table 2).

table 2 DC maturity level by surface maturity markers. CD34- CD80 + CD83 + CD86 + to me CF + DR + DNA 1 99.5 94 5 3,1 13.7 CF + DR + DNA 2 99 4.8 0.7 1,5 13 CF + DR 99 1.7 0.5 5.8 13.5 DR one hundred 2 - 1,1 19.5 CF one hundred 93 - 2,5 12,2 control (tumor) 99.5 7.8 - 2,4 17.7 intact 99 1,1 - 0.5 8

It was shown that the effect of cytostatics on mice with inoculated tumors leads to an increase in the number of CD80 + cells relative to the control group (7.8%) to 93 and 94% in the DR + CF + DNA (first mouse) and CF groups, respectively. The marker CD86 also showed an increase in its number in the groups DR + CF + DNA (first mouse) and DR + CF to 3.1 and 5.8%, respectively, compared with 2.5% in the control group. Markers CD80 and CD86 in tandem are surface differentiating clusters of antigen-presenting DCs that provide co-stimulatory signals for the activation and survival of T cells. In addition, treatment with cytostatics of CF and DR leads to an increase in the expression of the surface marker of mature CD83 CDs to 0.5% relative to the zero level in the control group. The CD83 surface marker regulates the development of CD4 + T cells. The expression level of surface CD83 reaches 5% with additional injections of DNA into animals (first mouse). The number of MHC class P molecules (I-Ad / I-Ed) is from 12.2 (CF) to 19.5% (DR) relative to 17.7% in the control mouse, i.e. overexpression of this surface marker is not observed.

The analysis of the number of perforin-containing CD8 + cytotoxic lymphocytes was also carried out in a suspension of cells from all organs (table 3).

Table 3 The number of CD8 + perforin-containing T cells. CD8a + perforin cf + dr + dna 1 8.4 36 Cf + DR + DNA 2 16.5 52.8 CF + DR 4.9 9.6 DR 5.2 23.9 CF 6.5 24.9 control (tumor) 6.6 21.7 intact 13,4 59.3

It turned out that injections of the DNA preparation against the background of injections of cytostatics CF and DR lead to an increase in the number of CD8 + cytotoxic lymphocytes to 8.4 and 16.5 (first and second mice) relative to the control (6.6%), increasing the level of intracellular, toxic to Perforin protein tumor cells up to 36 and 52.8%, respectively, against 21.7% in the control. This may indicate that injections of the DNA preparation against the background of pretreatment of CF and DR lead to an increase in the number of CD8 + perforin-containing cytotoxic lymphocytes toxic to tumor cells.

Table 4 The effect of intact, LPS, and DNA-treated DCs of healthy donors on the induction of CD8 + perforin + T cells in SCR. Donor number DK The content of CD8 + perforin + T cells in intact MNCs The content of CD8 + perforin + T cells in SCR induced in the presence of intact, LPS and DNA-activated DC 0 LPS DNA one 3.8 13 13 fifteen 2 3.8 10 12 fourteen 3 3.8 fourteen fourteen 12 four 3.8 12 10 eighteen 5 2,5 7.5 7.5 10 6 2,5 5 7.5 7.5 7 2,5 fifteen 12.5 22.5 Average 3.2 ± 0.26 10.9 ± 1.4 10.9 ± 1.0 14.1 ± 1.9

As follows from the data obtained, freshly isolated donor MNCs were characterized by a low content of CB8 + perforin + T cells (Fig. 7, table 4). Immature IFN-DK (allogeneic), when co-cultured with MNCs in SCR, induced a significant 3-fold increase in the number of cytotoxic T lymphocytes. Moreover, the activation of LPS DCs did not enhance the ability of DCs to induce cytotoxic T cells. At the same time, the growth of cytotoxic T cells in the presence of DNA-treated DC was higher than in the presence of intact DC. Thus, the Panagen preparation enhanced the ability of IFN-induced DCs to generate cytotoxic T-lymphocytes in SCR.

It was also found that in an in vitro system immunocompetent cells produce a significant amount of cytokines, which in principle can stimulate HSC proliferation [38]. Nevertheless, in cancer patients with breast cancer (nosology taken for research), overproduction of the entire palette of cytokines is found. This is due to the developing oncological process and inflammatory reactions of the body to dying malignant tissue. In this regard, the likelihood that stimulation of proliferation of bone marrow precursors is associated with an increase in the concentration of cytokines inducing HSC proliferation is less acceptable than the possibility of direct activation of HSC fission by double-stranded ends of HSC fragments of exogenous DNA internalized in the nuclear space. Either the concentration of the cytokine secreted by the cell is not important, but the amount of the intracellular cytokine of the immunocompetent cell that will be secreted in the immediate vicinity of the target cell is important.

Based on the above information, a method of treating cancer is as follows.

A method of treating oncological diseases is based on the use of cytostatic group preparations in breast cancer treatment regimens of the II-IV stage, characterized in that in addition to cyclophosphamide (500 mg / m ² ) and doxorubicin (50 mg / m) after a single chemotherapy cycle of intravenous administration ² ) (AC scheme) or cyclophosphamide (500 mg / m ² ), doxorubicin (50 mg / m ² ) and fluorouracil (500 mg / m ² ) (FAC scheme), the patient is prescribed a preparation based on Panagen exogenous human DNA (LSR No. 004429 / 08 dated 06/09/2008) in the form of tablets coated with a gastroenter 30 mg / day in a uniform dose during the active period of the day, which is one tablet of Panagen six times a day (every two hours) with the start of taking the drug immediately after chemotherapy in the amount of 3 tablets for 6 hours, a break in taking the drug, resuming it after 42 hours, that is, 48 hours after chemotherapy (day 3), continuing to take the drug for 17 days to 20 days after chemotherapy (inclusive), and continuing taking the drug according to the specified scheme until the end of all chemotherapy courses.

The use of drugs of the cytostatic group when they are synergistically combined with Panagen according to this scheme is a new strategy for the treatment of breast cancer, the use of which leads to the simultaneous manifestation of three types of therapeutic effects within the same chemotherapy course; to preservation and stimulation of granulocyte-macrophage and lymphocytic hematopoietic sprouts, which compensates for the negative leukoreducing effect of cytostatics, to preservation of innate anti-cancer immunity at the level prior to cytostatic therapy and the induction of anti-cancer adaptive immunity characterized by an increase in the peripheral blood of patients with high cytotoxic CD8 + lymphocytes with a high content of lymphocytes with high content of lymphocytes.

The strategy of using the combination of XT according to the AC and FAC regimens for the treatment of stage II-IV breast cancer was applied as part of the study of the Panagen drug in phase II clinical trials.

The following is experimental clinical evidence of the simultaneous manifestation of two therapeutic properties of the Panagen drug when used in the described strategy in combination with the cytostatics cyclophosphamide and doxorubicin in the treatment of stage II-IV breast cancer.

An example implementation of the method.

Used techniques.

The study involved patients treated at the Novosibirsk Cancer Center for stage II-IV breast cancer using a single intravenous injection of cytostatics cyclophosphamide (500 mg / m ² ), doxorubicin (50 mg / m ² ) and fluorouracil (500 mg / m ² ). Patients received an exogenous Panagen DNA preparation in the form of tablets coated with a gastroenteric coating at a dose of 30 mg / day with a uniform dose during the active period of the day, which will be one Panagen tablet six times a day (every two hours) with the start of the drug immediately immediately after XT in the amount of 3 tablets for 6 hours, a break in taking the drug, resuming it after 42 hours, that is, 48 hours after the XT (day 3) and continuing for 17 days to 20 days after le XT (inclusive) using the indicated scheme for 3 consecutive XT according to AC and FAC schemes.

The study used the dosage form of Panagen in the form of tablets coated with a gastroenteric coating. The tablet disintegrates in the intestines with a neutral pH value. The delivery of exogenous DNA to the bone marrow occurs through the absorption system of the small intestine. Along with absorption into the blood, exogenous DNA fragments are captured by immunocompetent cells of Peyer's intestinal plaques and other immune organs.

The study of Panagen was placebo-controlled. All patients who were in the placebo group received full standard treatment according to the requirements of the Ministry of Health and Social Development of the Russian Federation. Against the background of XT according to the scheme: cyclophosphamide (500 mg / m ² ), doxorubicin (50 mg / m ² ) and fluorouracil (500 mg / m ² ), patients in this group took a placebo one tablet six times a day, with a fractional uniform dose of during the active period of the day (one tablet every two hours). Placebo was started immediately after XT in the amount of 3 tablets for 6 hours, that is, one tablet every two hours. After that, patients stopped taking the placebo, resumed it after 42 hours, that is, 48 hours after the XT (day 3) and continued for 17 days to 20 days after the XT (inclusive).

In the XT 1 cycle, blood samples for determining the absolute number of leukocytes and neutrophils were collected 1-3 days before XT, 7 (± 1), 14 (± 2), and 21 (± 1) days after XT. Blood samples to determine the number of CD34 + cells will be collected 1-3 days before XT and then on day 7 (± 1) and day 21 (± 1) after XT. In cycle 2, blood samples to determine the absolute number of leukocytes and neutrophils were collected on 28 (± 1), 35 (± 2), and 42 (± 1) days after XT. Blood samples to determine the number of CD34 + cells were collected only on day 42 (± 1) after XT. In cycle 3, blood samples to determine the absolute number of leukocytes and neutrophils were collected on 49 (± 1), 56 (± 2), and 63 (± 1) days after XT. Blood samples to determine the number of CD34 + cells were collected only on day 63 (± 1) after XT.

Safety analysis (physical examination, basic vital signs) was carried out within 1-3 days before the start of XT in each cycle and 14 (± 2) days after the XT. Patients were observed for adverse events and concomitant medications during the study.

Tests of the end of the study were performed on 21 (± 2) days after the 3rd XT cycle. Biochemical blood tests are carried out 1-3 days before the start of XT and 21 (± 2) days after XT (Fig. 8).

XT was repeated every 3 weeks (if no break was required). To start XT on day 1 of the next cycle (day 22 of the previous cycle), the patient must have leukocytes ≥3.0 × 10 ⁹ / L, neutrophils ≥1.5 × 10 ⁹ / L, and platelet count ≥100 × 10 ⁹ / L . A delay in the administration of an XT dose of up to 1 week is acceptable.

During the study of various parameters of hematopoiesis, it was revealed that some patients responded to the effect of the drug, and some did not respond to the use of the drug Panagen. In connection with this division of patients for each evaluation parameter, the entire sample was divided by this criterion, that is, 2 groups were formed: “responding” and “not responding”.

Reliability assessment was carried out by the Mann-Whitney U-test. Parameters were evaluated both in absolute and in relative values. This was due to the scatter in the absolute numbers of the compared parameters for each individual patient. Two options for relative comparison were chosen.

1) All indicators were compared with respect to the "0" point - the point of analysis of the parameters before therapy. The relative figure was taken as a fraction (in%) of the initial value for each measurement point. Such a comparison made it possible to evaluate the dynamics of the recovery of the measured parameter with respect to its state before XT. That is, to evaluate the ability of the drug to compensate for the negative effect of cytostatics and characterize its supporting and restorative effect on the hematopoietic system over the course of XT courses.

2) 14 days after the administration of cytostatics, the study showed that they are the point of maximum inhibition of hematopoiesis (minimum values for blood cells) for patients receiving the drug Panagen. Also, as the results of pilot studies show, by the 14th day after the onset of XT, the effect of exposure to the drug (tablet form of Panagen) begins to appear. In this regard, all indicators of the research protocol were compared with indicators 14 days after the first course of XT. Relative figures were also taken as a percentage (in%) of the parameter indicator on the 14th control day. Such an analysis allowed: a) to conduct a relative assessment of the tread and restorative effect of the drug on hematopoiesis, regardless of the initial state of the patient’s hematopoietic system during several XT; b) compare the ability of the drug to maintain hematopoietic potential on the most critical days (14 days after the administration of cytostatics) of each subsequent XT, when there is the greatest likelihood of developing febrile neutropenia.

3) Patients were divided into 3 groups: “responding” to the action of the drug, “not responding” to the effect of the drug and “Placebo”.

Consider the characteristics of the main indicators of hematopoiesis in patients with breast cancer who received Panagen and placebo during the course of XT, in addition, we characterize the effect on the white blood sprout. Let us evaluate the dynamics of the content of various forms of leukocytes in the peripheral blood of patients participating in the study.

1. Evaluation of the number of leukocytes in the control point during the course of three courses of XT.

Evaluation of stimulation of leukocyte proliferation was carried out by comparing the number of leukocytes detected in the peripheral blood at control points in patients receiving Panagen and placebo. Both absolute and relative numbers were compared. With this comparison, it was supposed to determine the degree of influence of the drug on the stimulation of leukopoiesis in general against the background of the action of the cytostatic agent along the XT cycles. It was necessary to determine how the potential of progenitors of the leukocyte germ changes with the increasing negative impact of cytostatic treatment.

The dynamics of the content of the absolute number of leukocytes in the peripheral blood of patients at control points was evaluated (table 5). As follows from the analysis of the obtained data, the maximum leukopenia is detected on the 14th day after the administration of cytostatics (Fig.9) in all XT courses.

Table 5 The number of leukocytes (× 10 ⁹ / l) in patients receiving Panagen and placebo, in the difference control points during 3 courses of XT. 0 7 day 14 day 21 day After 1 HT After 2 XT After 3 HT After 1 HT After 2 XT After 3 HT After 1 HT After 2 XT After 3 HT Panagen Awa 7.7 5.1 0.9 1,5 10.3 6.0 B-va 7.0 5,0 6.1 7.5 7.8 B-ko 4.8 4.0 2,3 4.0 1.9 5.8 6.0 5.2 B-r 8.8 3.0 2,4 3.0 2.0 2,4 2,3 5,6 5.1 4.8 In 9.6 4.2 4.2 15.0 3.0 2.9 2,8 6.7 4.9 4.3 G-ko 8.2 4.0 3.8 4.7 2.0 2.7 3,5 6.1 7.6 6.3 G-va 6.5 3.0 3.6 3.4 2.1 5.1 2.2 5,0 11.1 5,0 Ev 6.9 3.6 3.6 3.4 1,5 3.4 1.3 6.9 6.6 4.4 Eve 8.6 2,3 5.2 3.9 2.7 2,8 3,1 10.9 8.8 9.0 Willow 9.0 5.3 5.2 5,6 2.1 2.7 3.4 7.1 7.0 5,0 Cow 7.2 9.7 6.5 3.6 4.4 2.7 3.2 7.0 4.9 M-ko 6.6 5.5 3.9 3.6 3.8 M-va 8.1 5,4 5,4 2.9 2,5 3.3 7.4 5.9 5.1 Md 3,7 2,5 2.7 3,1 2,3 2.7 3.4 4.2 3.3 M-on 6.5 4.3 4.3 2.2 1.9 3.0 6.0 7.2 5.8 P-on 5.8 4.1 1,6 Peninsula 4,5 2.9 2,8 3.3 5.1 median 7.1 4.2 4.0 4.0 2.2 2.7 2,8 6.1 6.3 5,0 Placebo Two 3.2 2.9 4.3 2,3 2.1 D 6.2 5.2 5.1 2.2 2,4 2,4 5,0 6.2 3.9 Quality 7.6 4.6 3,5 4.1 2.2 2.1 2,4 8.1 5,0 4.3 K-va 6.7 5,0 4.3 3.6 3.2 1.3 4.4 4.6 3.6 Sh-va 8.3 3.2 3.4 1,2 2.9 2,4 3.6 median 6.7 4.6 3,5 4.6 2.2 2.7 2,4 4.4 4.6 3.8

An assessment was made of changes in the conditional content of leukocytes with a breakdown into the groups “responding” and “not responding” to the effect of the drug in relation to the indicators of the first XT (tables 6, 7) and relative to the initial level (table 8). On the 21st day after the 3 XT, there is a difference in the number of peripheral blood leukocytes between the Placebo and Panagen-responding groups with a p _u <0.01 reliability (Fig. 10). Moreover, the number of leukocytes in the placebo group does not reach the level detected on the same day after 1 XT (100%), while in the group of patients responding to the drug, this figure exceeds 100%.

Table 6 The conditional leukocyte count in% relative to the level after 1 XT in patients receiving Panagen and placebo on days 7 and 14 after 2 and 3 XT. 7 day 14 day After 2 XT After 3 HT After 2 XT After 3 XT Panagen Awa 17.6 B-ko 83.3 173.9 82.6 B-r 80.0 100.0 120.0 115.0 In 100.0 357.1 96.7 93.3 G-ko 95.0 117.5 135.0 175.0 G-va 120.0 113.3 242.9 104.8 Ev 100.0 94.4 226.7 86.7 Eve 226.1 169.6 103.7 114.8 Willow 98.1 105.7 128.6 161.9 Cow 122,2 75.0 M-va 100.0 86.2 113.8 Md 108,0 124.0 117.4 M-on 100.0 86.4 136.4 median 100.0 105.7 121.1 113.8 Placebo D 98.1 109.1 109.1 Quality 76.1 89.1 95.5 109.1 K-va 86.0 88.9 36.1 Sh-va 106.3 241.7 median 86.0 93.6 102.3 109.1

Table 7 The conditional leukocyte count in% relative to the level after 1 XT in patients receiving Panagen and placebo on day 21 after 2 and 3 XT. After 2 XT After 3 HT Panagen - answering B-va 123.0 127.9 G-ko 124.6 103.3 G-va 222.0 100.0 Cow 218.8 153.1 Md 123.5 97.1 M-on 120.0 96.7 Peninsula 117.9 182.1 median 123.5 103.3 Panagen - not responding M-ko 92.3 97.4 B-ko 103,4 89.7 Awa 58.3 B-r 91.1 85.7 In 73.1 64,2 Ev 95.7 63.8 Eve 80.7 82.6 Willow 98.6 70,4 M-va 79.7 68.9 median 91.1 76.5 Placebo Two 53.5 48.8 D 124.0 78.0 Quality 61.7 53.1 K-va 104.5 81.8 Sh-va 150.0 median 104.5 65.5

Table 8 The conditional leukocyte content in% relative to the initial level in patients receiving Panagen and placebo at various control points during 3 courses of chemotherapy. 7 day 14 day 21 day After 1 HT After 2 XT After 3 ht After 1XT After 2 XT After 3 HT After 1XT After 2 XT After 3 HT Panagen Awa 66,2 11.7 19.5 133.8 77.9 B-va 71,4 87.1 107.1 111.4 B-r 34.1 27.3 34.1 22.7 27.3 26.1 63.6 58.0 54.5 In 43.8 43.8 156.3 31.3 30,2 29.2 69.8 51.0 44.8 G-ko 48.8 46.3 57.3 24.4 32.9 42.7 74,4 92.7 76.8 G-va 46.2 55,4 52.3 32,3 78.5 33.8 76.9 170.8 76.9 Ev 52,2 52,2 49.3 21.7 49.3 18.8 100.0 95.7 63.8 Eve 26.7 60.5 45.3 31,4 32.6 36.0 126.7 102.3 104.7 Willow 58.9 57.8 62,2 23.3 30,0 37.8 78.9 77.8 55.6 Cow 134.7 90.3 50,0 61.1 37.5 44,4 97.2 68.1 M-ko 83.3 59.1 54.5 57.6 M-va 66.7 66.7 35.8 30.9 40.7 91.4 72.8 63.0 Md 67.6 73.0 83.8 62,2 73.0 91.9 113.5 89.2 M-on 66,2 66,2 33.8 29.2 46.2 92.3 110.8 89.2 P-on 70.7 27.6 Peninsula 64,4 62,2 73.3 113.3 median 64,4 56.6 57.3 31.3 31.7 36.8 78.9 92.7 72,4 Placebo Two 90.6 134.4 71.9 65.6 D 83.9 82.3 35.5 38.7 38.7 80.6 100.0 62.9 Quality 60.5 46.1 53.9 28.9 27.6 31.6 106.6 65.8 56.6 K-va 74.6 64,2 53.7 47.8 19,4 65.7 68.7 53.7 Sh-va 38.6 41.0 14.5 34.9 28.9 43,4 median 74.6 46.1 68.1 32,2 36.8 31.6 80.6 68.7 59.7

In the process of analyzing the results, the following fact was discovered. Some patients included in the groups of “responding” and “not responding” to the action of the Panagen drug change places in two consecutive control points (for example, 14 and 21 days after XT). That is, in patients who responded by increasing the number of leukocytes in peripheral blood on the 14th day after injection of cytostatics, the number of leukocytes by 20 days after administration of cytostatics was at or less than the corresponding control point after 1 XT, taken as 100%. Conversely, in patients who had not yet responded to the action of the drug on day 14, sensitivity to Panagen was induced by day 20. Moreover, the number of leukocytes in the peripheral blood of patients in the group responding to the action of the drug Panagen at the control point 21 days after XT is significantly higher than in the placebo group. It can be assumed that the proliferative activity of committed unipotent leukocyte precursors is activated by the drug in time-sensitive “susceptibility windows”. This leads to a shift in the proliferation time, which is reflected in the number of leukocytes recorded in the peripheral blood of patients.

The number of patients examined in the course of 2 XT on day 14 is 12 people, of which “responding” - 9, “not responding” - 3. The number of patients examined in 2 XT on day 14 is 16 people, of which “responding” ”- 8,“ non-responders ”- 8. Of these, one person from the Panagen-non-responders group moved to the Panagen-non-responders group and 4 people from the non-responders group went to the responders group (FIG. 11 table 9).

The number of patients examined in the course of 3 XT on the 14th day - 11 people, of which “responding” - 7, “not responding” - 4. The number of patients examined in the course of 3 XT on 21 days - 15 people, of which “ respondents ”- 5,“ non-responders ”- 10. Of these, 3 people from the Panagen-responders group moved to the Panagen-non-responders group. From the group “not responding” to the group “responding” one person moved (Fig. 12, table 10). Moreover, on the 21st day after 3 XT, the number of leukocytes in the placebo group does not reach the level detected on the same day after 1 XT (100%), while in the group of patients responding to the drug, this indicator reaches 130%.

That is, it can be said that the group of “responding” patients is much larger, which is associated with the spread in the response time to the drug for each individual patient. That is, those patients who switched to the group of “non-responders” already responded with a rise in leukocytes a few days earlier, that is, the drug performed its leukostimulating function.

Table 9 The conditional leukocyte count in% relative to the level after 1 XT in patients receiving Panagen and placebo on days 14 and 21 after 2 XT. 14 day 21 day Panagen - answering B-ko 173.9 B-va 123.0 B-r 120.0 B-ko 103,4 G-ko 135.0 G-ko 124.6 G-va 242.9 G-va 222.0 Ev 226.7 Cow 218.8 Eve 103.7 Md 123.5 Willow 128.6 M-on 120.0 Cow 122,2 Peninsula 117.9 Md 117.4 median 128.6 median 123,2 Panagen - not responding In 96.7 M-ko 92.3 M-va 86.2 Awa 58.3 M-on 86.4 B-r 91.1 In 73.1 Ev 95.7 Eve 80.7 Willow 98.6 M-va 79.7 median 86.4 median 85.9 Placebo Two Two 53.5 D 109.1 D 124.0 Quality 95.5 Quality 61.7 K-va 88.9 K-va 104.5 Sh-va 241.7 Sh-va 150.0 median 102.3 median 104.5

Table 10 The conditional leukocyte count in% relative to the level after 1 XT in patients receiving Panagen and placebo on days 14 and 21 after 3 XT. 14 day 21 day Panagen responders B-r 115.0 B-va 127.9 G-ko 175.0 G-ko 103.3 G-va 104.8 G-va 100.0 M-va 113.8 Cow 153.1 Eve 114.8 Peninsula 182.1 Willow 161.9 M-on 136.4 median 115.0 median 127.9 Panagen non-responding B-ko 82.6 B-ko 89.7 Cow 75.0 Md 97.1 Ev 86.7 M-on 96.7 In 93.3 M-ko 97.4 B-r 85.7 In 64,2 Ev 63.8 Eve 82.6 Willow 70,4 M-va 68.9 median 84.6 median 84.1 Placebo Two Two 48.8 D 109.1 D 78.0 Quality 109.1 Quality 53.1 K-va 36.1 K-va 81.8 median 109.1 median 65.5

2. Let us estimate the number of neutrophils at control points during three XT courses.

The number of neutrophils in the peripheral blood of patients was determined at all XT control points (table 11).

Stimulation of the neutrophilic hemopoietic leaf stimulation was carried out by comparing the number of neutrophils detected in the peripheral blood at control points in patients receiving placebo and Panagen. Relative numbers were compared. With such a comparison, it was supposed to determine the degree of influence of the drug on the stimulation of neutropoiesis against the background of the action of the cytostatic agent along the XT cycles. It was necessary to determine how the potential of neutrophil progenitors changes with the increasing negative impact of cytostatic treatment.

The dynamics of the content of neutrophils in the peripheral blood of patients at the control points after 2 and 3 XT was evaluated (Fig.13, 14, table 12). In this case, the value at the control point 1 XT was taken as 100%. The remaining values were taken as a percentage (%) of this figure.

Table 12 Conditional neutrophil content in% relative to the level after 1 XT in patients receiving Panagen and placebo on days 14 and 21 after 2 and 3 XT. 14 day 21 day After 2 XT After 3 HT After 2 XT After 3 HT Panagen responders B-ko 195.3 37.7 B-va 133.0 153.0 B-r 282.0 356.5 B-ko 133.8 103.6 In 108,4 107.5 G-ko 120.8 109.6 Md 113.1 G-va 215.9 90,4 G-va 185.5 84,4 Ev 122.5 73.8 Willow 178.7 205.3 Cow 231.3 28,4 Cow 177.5 117.9 Md 159.5 112.9 M-va 139.6 173.4 M-on 146.2 109.0 Peninsula 105.5 206.1 median 178.1 117.9 median 133.8 109.0 Panagen non-responding G-ko 40.1 99.3 Awa 77.3 Eve 87.9 81.7 B-r 81.8 93.0 M-on 63.6 59.8 In 74.5 32.1 Eve 84.2 73.1 Willow 72.1 31.5 M-ko 84.8 89.5 M-va 85.3 67.9 median 63.6 81.7 median 81.8 70.5 Placebo Two Two 40.3 31.6 D 89.8 89.8 D 116.4 86.4 Quality 130,4 143.7 Quality 53.0 47.3 K-va 90.3 33,2 K-va 118.4 94.2 Sh-va 295.4 Sh-va 164.0 median 110,4 89.8 median 116.4 66.8

The effect of preserving the number of neutrophils in each group as they progressed along XT cycles at control points 14 and 21 days after cytostatic injection was also evaluated (Fig. 15, 16). In this case, the group indices after each XT were compared. For 100%, the value of the indicator determined in the first XT was taken. A comparison of the graphs suggests that the drug on day 14 of each subsequent XT continues to maintain the number of neutrophils in the blood of patients. With a similar comparison on day 21 after 3 XT in patients taking the drug, there is still an excess of the indicator over the starting point. Whereas in the placebo group there was a sharp decrease in the number of neutrophils in the blood (Fig. 16).

A comparison of the relative number of neutrophils with respect to the starting point will indicate the degree of restoration of the number of neutrophils to the initial level determined before cytostatic treatment (Fig. 17, table 13). The comparison shows that on day 14 during the execution of XT there are no differences. On the 21st day, the Placebo and Panagen - not responding groups showed a tendency to a gradual decrease in the number of peripheral neutrophils. In the Panagen-responders group, there is a tendency to stabilize the level of neutrophils in peripheral blood.

Table 13 The conditional neutrophil content in% relative to the initial level in patients receiving Panagen and placebo at various control points during 3 XT courses. 14 day 21 day After 1 HT After 2 XT After 3 HT After 1 HT After 2 XT After 3 HT Panagen responders B-r 7.6 21,4 27.0 B-va 69.9 93.0 107.0 In 15.4 16.7 16.5 B-ko Md 54.3 61,4 G-ko 74,4 89.8 81.6 G-va 35.8 66,4 30,2 G-va 86.4 186.5 78.1 Willow 12.6 22.5 25.8 Ev 100.0 122.5 73.8 Cow 28.0 49.7 33.0 Cow 41.5 95.9 11.8 M-va 24.7 34,4 42.7 Md 80.2 127.9 90.6 M-on 74.7 109.1 81.4 Peninsula 47.3 49.9 97.5 median 24.7 34,4 28.6 median 74.5 102.5 81.5 Panagen non-responding G-ko 27.8 11.1 27.6 Awa 98.2 75.9 Eve 25.0 22.0 20.5 B-r 62.6 51,2 58.2 M-on 28,4 18.1 17.0 In 56.3 41.9 18.1 Eve 119.9 100.9 87.7 Willow 69.5 50.1 21.9 M-ko 41,4 35.1 37.0 M-va 106.3 90.8 72.3 median 27.8 18.1 20.5 median 69.5 51,2 47.6 Placebo Two Two 153.9 62.0 48.7 D 39.6 35.5 35.5 D 85.9 100.0 74,2 Quality 15.6 20,4 22.4 Quality 89.8 47.6 42,4 K-va 47.5 42.9 15.7 K-va 50,4 59.7 47.5 Sh-va 6.8 20,0 Sh-va 16.1 26.5 median 27.6 27.9 22.4 median 85.9 59.7 48.1

In the process of analyzing the results, the following fact was discovered. Some patients included in the groups of “responding” and “not responding” to the action of the Panagen drug in two successive control points (for example, 14 and 21 days after XT) are interchanged. That is, in patients who responded by increasing the number of neutrophils in peripheral blood on the 14th day after the injection of cytostatics, the number of neutrophils by 21 days after the administration of cytostatics was at or less than the corresponding control point after 1 XT, taken as 100%. Conversely, in patients who had not yet responded to the action of the drug on day 14, sensitivity to Panagen was induced by day 21. It can be assumed that the proliferative activity of committed unipotent precursors of bone marrow resident neutrophils is activated by the drug in time-sensitive “susceptibility windows”. This leads to a shift in the proliferation time, which is reflected in the number of neutrophils recorded in the peripheral blood of patients.

The number of patients examined during 2 XT on the 14th day is 11 people, of which “responding” - 8, “not responding” - 3. The number of patients examined in the course of 2 XT on 21 days - 15 people, of which “ respondents "- 9," non-responders "- 7. Of these, three people from the" Panagen - responders "group moved to the" Panagen - non-responders "group, and 1 person from the" non-responders "group went to the" responders "group (FIG. .18. Table 14).

The number of patients examined during 3 XT on day 14 was 10 people, of which “responding” - 5, and “not responding” - 5. The number of patients examined during 3 XT on day 21 was 15 people, of which “responding” ”- 6,“ non-responders ”- 9. Of these, five people from the“ Panagen-responding ”group moved to the“ Panagen-non-responding ”group, and 3 people from the“ non-responding ”group moved to the“ responding ”group (Fig. 19. table 15).

That is, again, we can say that the group of “responding” patients is much larger, which is associated with a spread in the response time to the drug.

Table 14 The conditional neutrophil content in% relative to the level after 1 XT in patients receiving Panagen and placebo on days 14 and 21 after 2 XT. 14 day 21 day Panagen responders B-ko 195.3 B-va 133.0 B-r 282.0 B-ko 133.8 In 108,4 G-ko 120.8 G-va 185.5 G-va 215.9 Willow 178.7 Ev 122.5 Md 113.1 Cow 231.3 Cow 177.5 Md 159.5 M-va 139.6 M-on 146.2 Peninsula 105.5 median 178.1 median 133.8 Panagen non-responding G-ko 40.1 Awa 77.3 Eve 87.9 B-r 81.8 M-on 63.6 In 74.5 Eve 84.2 Willow 72.1 M-ko 84.8 M-va 85.3 median 63.6 median 81.8 Placebo Two Two 40.3 D 89.8 D 116.4 Quality 130,4 Quality 53.0 K-va 90.3 K-va 118.4 Sh-va 295.4 Sh-va 164.0 median 110,4 median 116.4

Table 15 The conditional neutrophil content in% relative to the level after 1 XT in patients receiving Panagen and placebo on days 14 and 21 after 3 XT. 14 day 21 day Panagen responders B-r 356.5 B-ko 103.6 In 107.5 B-va 153.0 Willow 205.3 G-ko 109.6 Cow 117.9 Md 112.9 M-va 173.4 M-on 109.0 Peninsula 206.1 median 173.4 median 111.3 Panagen non-responding B-ko 37.7 B-r 93.0 G-ko 99.3 G-va 90,4 G-va 84,4 Ev 73.8 Eve 81.7 Cow 28,4 M-on 59.8 In 32.1 Eve 73.1 Willow 31.5 M-ko 89.5 M-va 67.9 median 81.7 median 73.1 Placebo Two 31.6 D 89.8 D 86.4 Quality 143.7 Quality 47.3 K-va 33,2 K-va 94.2 median 89.8 median 66.8

It can be noted that the group of "responders" to the drug after all XT courses makes up one third of the total number of patients taking the Panagen drug, and in terms of the relative neutrophil content on day 21 after 3 XT significantly (p _u <0.01) differs from the group "Panagen - not responding," and from the placebo group.

It was found that the maximum number of neutropenia of all degrees occurs on the 14th day after the introduction of cytostatics (Fig.20, table 16). The most frequent was a decrease in the level of neutrophils to the level of 1.0-1.5 × 10 ⁹ / L, which corresponds to neutropenia of the II degree. When comparing the frequencies of occurrence of grade II neutropenia in the Panagen and Placebo groups, attention is drawn to the preservation of the frequency of occurrence in the Panagen group for 3 XT, during which the study was conducted, and the progressive increase in the frequency of neutropenia in the Placebo group. By the third XT, the number of patients with symptoms of neutropenia due to XT in the placebo group increased by 2 times compared with the Panagen group (Fig.21).

Despite the fact that in several patients the neutrophil counts fell to the level of neutropenia of the fourth degree, after repeated measurements in the next 3 days, the neutrophil level was restored to above critical levels and at the control point 21 days before the next XT, the number of neutrophils reached the level allowing the next course XT (FIG. 22, table 17).

Table 17 Dynamics of recovery of neutrophil count after stage IV neutropenia in the Panagen and Placebo groups. Day after XT fourteen 16-19 21 Panagen Awa 0.27 3.27 4.62 B-r 0.40 0.91 3.30 Ev 0.34 0.62 2.90 B-ko 0.49 3.48 average 0.38 1,60 3,58 Placebo Sh-va 0.43 1,03

Estimation of the number of monocytes at control points during the course of three XT courses.

Evaluation of the effect of the drug on the monocytic blood sprout was carried out according to a similar principle used to assess white blood cells and neutrophils. Firstly, a general comparison was made between the Placebo and Panagen groups of the absolute number of monocytes in the blood of patients participating in the trial. Such a comparison made it possible to establish that, like previous blood cells, monocytes repeat the dynamics of a decrease in the number of cells in peripheral blood in response to exposure to XT. Day 14 is a control point at which the number of monocytes in peripheral blood is minimal (Fig. 23, table 18).

Next, a comparison was made of the conditional content of monocytes in the “Placebo”, “Panagen - responding”, “Panagen - not responding” groups in relation to the indicators after 1 XT, which were taken as 100% in the control points after 2 and 3 XT (Fig. 24 25, table 19). A comparison was also made of the values at the control points within each group during XT (Fig. 25).

As in the previous sections, we estimated the conditional values of the monocyte content in each group at the second and third year of XT at control points 14 and 21 days after the administration of cytostatic relative to the parameter value after 1 XT (Figs. 26, 27, tables 20, 21). The Panagen-responding and Panagen-non-responding groups were formed independently for each control point. The results obtained indicate that the Panagen preparation allows maintaining the high number of monocytes in the peripheral blood of patients responding to the drug on critical days (14 days) after chemotherapy treatment.

Table 20 The conditional content of monocytes in% relative to the level after 1 XT in patients receiving Panagen and placebo on days 14 and 21 after 2 XT. 14 day 21 day Panagen responders B-ko 173.9 In 131.6 B-r 300,0 G-ko 373.8 In 483.3 G-va 333.0 G-va 485.7 Willow 147.9 Willow 180.0 Cow 437.5 M-on 863.6 M-ko 166.2 M-va 239.2 Peninsula 101.8 median 391.7 median 202.7 Panagen non-responding G-ko 90.0 Awa 87.4 Cow 43.7 B-ko 51.7 Eve 83.0 B-va 85.1 M-va 34.5 Eve 80.7 Md 78.3 B-r 91.1 Ev 31.9 M-on 36.0 Md 22.5 median 78.3 median 66,2 Placebo Two Two 192.6 D 109.1 D 24.8 Quality 152.7 Quality 49.4 K-va 33.3 K-va 418.2 Sh-va Sh-va 450,0 median 109.1 median 192.6

Table 21 The conditional content of monocytes in% relative to the level after 1 XT in patients receiving Panagen and placebo on days 14 and 21 after 3 XT. 14 day 21 day Panagen responders In 513.3 B-ko 537.9 G-ko 641.7 In 166.9 G-va 838.1 G-ko 464.8 Eve 229.6 G-va 200,0 Willow 291.4 Eve 275.2 M-on 136.4 Willow 223.0 Cow 842.2 M-ko 188.1 M-va 137.8 Peninsula 173.9 median 402.4 median 211.5 Panagen non-responding B-ko 41.3 B-va 49.2 B-r 57.5 B-r 19.0 Cow 10.7 Ev 63.8 M-va 68.3 M-on 38.7 Md 8.8 median 49.4 median 38.7 Placebo Two 232.0 D 109.1 D 109,2 Quality 218.2 Quality 116.8 K-va 18.1 K-va 327.3 median 109.1 median 174.4

Let us evaluate the dynamics of the content of the total pool of lymphocytes in the peripheral blood of patients participating in the study.

Lymphocytic, as well as the mononuclear fraction of the periphery, is the most important indicator of the immunocompetent state of the body of a patient with an oncological disease. Antigen-presenting cells of macrophages and DCs develop from the mononuclear fraction. The lymphocytic fraction consists of the main effectors of the immune system of T and B lymphocytes.

During the study, an assessment was made of the number of total pool of lymphocytes during XT (table 22).

Assessment of the content of lymphocytes was carried out according to the procedure described above. The analysis results indicate that at the control point after 3 XT the number of lymphocytes in the blood of patients is significantly higher in the Panagen group (p _u <0.05), without division into responders / non-responders than in the Placebo group (Fig. 28).

Also, an assessment of the differences in the parameters at the control point 21 days after 3 XT when divided into Panagen-responding, Panagen-not responding, and Placebo groups indicates that in the Panagen-responding group with a confidence of p _u <00 , 01 the number of lymphocytes is greater than in the placebo group. And with a probability of p _u <0.05, the number of lymphocytes in the Panagen-responding group is greater than in the Placebo group at the control point 21 days after 2 XT (Fig. 29, table 23). The results obtained indicate that the Panagen preparation contributes to the preservation and maintenance of peripheral lymphocytes when using intensive XT regimes.

Table 23 Conditional lymphocyte content in% relative to the level after 1 XT in patients receiving Panagen and placebo on days 14 and 21 after 2 and 3 XT. 14 day 21 day After 2 XT After 3 HT After 2 XT After 3 HT Panagen responders B-ko 140.0 46.3 B-r 108.1 88.4 M-on 49.4 220.8 Willow 159.3 127.3 Md 141.6 G-va 211.9 113.6 B-r 147.7 103,2 G-ko 116.8 71.0 Willow 82.5 116.1 Cow 164.1 142.2 Eve 129.6 153.1 Peninsula 139.6 172.6 G-va 401.6 133.0 B-va 139.7 110,4 G-ko 322.8 350,0 Cow 161.3 90.0 median 141.6 124.5 median 139.7 113.6 Panagen - not responding In 80.1 74,4 Ev 66,4 49.6 M-va 48.8 66.6 M-on 99,4 88.4 M-va 59.8 66.5 Md 79.9 88.5 In 67.4 98.0 Eve 68.3 85.7 Awa 23.0 M-ko 73,4 81.2 B-ko 67.2 47.1 median 64,4 70.5 median 67.4 83.5 Placebo Two 68.7 64.5 D 151.3 144.3 D 148.8 54.6 Quality 64.8 74.1 Quality 82.3 53.1 K-va 103.7 48.1 K-va 76.8 56.8 Sh-va 171.5 Sh-va 116.7 median 127.5 74.1 median 82.3 55.7

A comparison of the graphs suggests that the drug on the 21st day of each subsequent XT continues to maintain the number of lymphocytes in the blood of patients. In the placebo group, there is a significant reduction in the number of lymphocytes in the peripheral blood with each subsequent XT. With a similar comparison on day 14 after 3 XT in patients taking the drug, a significant excess of the indicator over the starting point is also observed. Whereas in the placebo group on the 21st day after 3 XT there was a sharp decrease in the number of lymphocytes in the peripheral blood with a reliability of p _u <0.01 (Fig. 30).

Let us characterize the effect on the red sprout of blood.

The impact of the Panagen preparation was assessed using several standard indicators reflecting the state of the red blood sprout during XT (table 24). As follows from the analysis, the drug Panagen does not have a significant effect on the red sprout of hematopoiesis. The only noted sign was an increase in platelet count by the 21st control day of each XT in several patients taking Panagen (no significant differences in the total sample compared with the placebo group were detected).

Table 24 Values of indicators of red blood germ in groups of patients receiving Panagen and placebo at various control points XT. An asterisk (*) indicates the average, significantly different from the norm. Red blood cells Norm - from 3.8 to 5.1 0 After 1 XT After 2 XT After 3 HT 7 fourteen 21 7 fourteen 21 7 fourteen 21 Panagen Ev 4,58 4.33 4.68 4.33 3.99 4.42 4.23 3.89 3.91 B-ko 4.36 3.98 4.11 4.16 3.94 3.89 3.99 M-on 4,51 5.07 4.43 4.83 4.25 4,57 4.18 3.97 P-on 4,5 3.84 3.17 M-va 4,57 4.13 4.36 4,56 3.96 3.66 4.26 4.05 4.09 Md 3.98 3.83 3.85 4,51 4.2 4.04 3.81 4.01 3.77 B-r four 3,51 3.2 3.25 3.16 3.08 3,1 3.22 3.13 2.83 Willow 5.83 4.29 4.02 4.18 3.79 3,7 4.08 3.93 3.78 3,58 Eve 3.88 3.91 3.69 4.14 4.12 3,58 3.57 3.23 3.66 3.74 In 4.06 3.81 3.75 3.94 3.37 3.35 3.88 4.17 3.68 3.87 Awa 4,53 4.73 4.4 3.34 3.78 2.71 2.68 G-ko 3.85 3.67 3,7 3.88 4.3 3.98 4.22 4.23 3.89 4.1 G-va 3.18 3.21 3.68 3.42 3.68 3.66 3.86 3.65 3.38 3.83 Cow 4.72 4.24 4.21 4.15 4.41 4.68 4.23 4.11 4.02 Peninsula 4.86 4,52 4.2 4.32 4,59 B-va 4.46 4.4 4.68 4.44 4.09 M-ko 5.05 4.74 4,5 4.44 4.31 average 4.40 4.16 3.88 4.22 3.91 3.78 4.09 3.80 3.68 3.91 Placebo D 3.89 3.89 3.44 4.12 3.91 4.09 4.15 3.83 3.88 K-va 4.33 4.05 4.29 4.38 4.03 3.74 4.27 3,7 4.08 Quality 4.46 4.18 3.18 4.32 3.27 3.46 3.92 3.65 3,51 3.31 Two 4.7 4.26 4.34 4.27 4.13 average 4.35 4.10 3.64 4.29 3.65 3.70 4.14 3.90 3.68 3.85 Platelets Norm - from 150 to 400 0 After 1 XT After 2 XT After 3 XT 7 fourteen 21 7 fourteen 21 7 fourteen 21 Panagen Ev 111 184 263 143 220 282 154 171 265 B-ko 186 172 288 265 255 152 261 M-on 227 228 183 294 170 231 150 318 P-on 155 205 M-va 281 240 347 541 274 190 488 219 344 Md 155 165 114 244 159 136 176 210 233 B-r 232 178 229 461 184 150 436 229 174 293 Willow 178 135 170 252 137 188 337 198 163 280 Eve 331 211 391 403 187 212 303 146 306 427 In 252 238 192 265 163 161 364 188 173 362 Awa 257 168 255 265 331 92 89 G-ko 254 216 255 175 191 142 221 181 161 202 G-va 530 330 299 212 288 117 324 270 181 350 Cow 381 258 327 215 298 359 246 194 350 Peninsula 179 180 203 229 190 B-va 298 300 240 440 M-ko 329 233 348 395 408 average 277.4 204.6 230.7 298.2 194.1 193.4 315.4 187.6 191.8 314.6 Placebo D 225 228 150 286 180 254 194 161 323 K-va 264 208 196 370 246 199 274 194 243 Quality 324 200 145 251 210 124 359 263 155 266 Two 172 193 315 165 283 average 246.3 207.3 163.7 305.5 228.0 167.7 263.0 228.5 170.0 278.8 Hemoglobin Norm - from 110 to 155 0 After 1 XT After 2 XT After 3 HT 7 fourteen 21 7 fourteen 21 7 fourteen 21 Panagen Ev 137 130 139 129 119 132 123 114 116 B-ko 128 120 124 126 110 110 116 118 M-on 101 119 106 124 116 128 120 117 P-on 132 120 97 M-va 124 106 117 125 103 95 116 114 106 Md 114 119 111 126 118 114 107 111 111 B-r 120 107 98 103 95 93 92 98 97 87 Willow 165 125 118 128 124 111 124 118 116 108 Eve 118 114 109 123 122 106 109 107 112 114 In 130 119 123 128 122 112 127 121 121 127 Awa 142 151 117 111 123 88 87 G-ko 106 102 102 107 116 108 117 119 112 113 G-va 93 97 111 107 115 109 112 112 108 111 Cow 136 127 127 134 131 139 130 131 124 Peninsula 144 134 124 135 146 B-va 138 144 146 141 130 M-ko 154 148 134 132 133 average 127.8 123.1 113.0 123.9 117.8 111.6 121.5 113.1 112.1 117.4 Placebo D 118 116 105 129 125 127 128 118 120 K-va 118 111 119 127 111 108 127 114 121 Quality 175 110 108 125 104 one hundred 112 102 101 107 Two 132 124 122 119 117 average 135.8 115.3 110.7 125.8 107.5 111.0 121.3 115.0 111.0 116.3 The average hemoglobin content in the red blood cell Norm - from 27 to 34 0 After 1 XT After 2 XT After 3 XT 7 fourteen 21 7 fourteen 21 7 fourteen 21 Panagen Ev 29.9 thirty 29.7 29.8 29.8 29.9 29.1 29.3 29.7 B-ko 29.4 30.1 30,2 30.3 27.8 29.8 29.6 M-on 22.4 23.5 23.9 25.7 27.3 28 28.7 29.5 P-on 31.3 M-va 27 25.7 26.8 27.4 25.9 26.1 27,2 28.1 26 Md 28.8 30.9 28 27.9 28.1 28,2 28.1 27.8 29.2 B-r 30,4 30.5 31.6 thirty 30,2 29.7 30,4 31 30.7 Willow 29.1 32,5 30.6 32,7 thirty 30,4 thirty 30.7 Eve 30,4 29.7 29.6 29.6 30.6 33.3 30.6 30.5 In 31,2 32.8 32.6 36,2 33.5 32,7 29th 32.9 32.8 Awa 33.3 32,5 32,4 32.6 G-ko 27 27,2 27.8 28.1 28.7 27.6 G-va thirty 29.6 29.8 29.1 30.6 32 29.1 Cow 29.9 30.1 29.7 29.8 30.9 32 Peninsula 29.6 29.6 31.3 31.8 B-va 30.9 32,7 31,2 31.8 31.8 M-ko 30.5 31,2 39.8 29.7 30.9 average 28.5 29.6 29.5 30.5 29.9 29.6 29.8 30,0 30.6 30,0 Placebo D 30.3 29.8 30,4 31.3 32 31.1 30.8 30.8 30.9 K-va 27,2 27.4 27.7 29th 27.5 28.8 29.7 30.8 29.7 Quality 27.7 28.3 29th 31.8 29.1 28.6 28 28.8 32,3 Two 28.1 29.1 28.1 27.9 28.3 average 28.5 28.5 28.8 29.4 29.7 30,0 29.3 29.4 30.1 30.3 Erythrocyte sedimentation rate Norm - up to 20 0 After 1 XT After 2 XT After 3 HT 7 fourteen 21 7 fourteen 21 7 fourteen 21 Panagen Ev 25 5 24 eighteen 44 39 27 33 23 B-ko 12 29th 36 42 35 thirty 39 fifteen M-on 22 12 35 25 35 40 5 54 P-on 6 5 10 M-va 10 twenty 10 24 twenty twenty 25 10 fifteen Md 7 four 10 3 6 7 fifteen 7 6 B-r 57 5 49 38 42 34 32 42 40 fifty Willow eleven fifteen twenty 24 eighteen 7 17 5 twenty 10 Eve 35 31 28 38 36 38 24 28 38 In 9 16 fourteen four 23 twenty twenty 10 8 7 Awa 16 36 G-ko 23 fourteen 10 twenty 23 35 thirty 22 12 eighteen G-va twenty 34 28 34 21 31 40 26 7 22 Cow 28 fourteen 24 28 23 eleven eleven 13 eleven average 20.3 16.1 20,2 25,4 22.1 27.8 * 28.5 * 19.0 24.0 19.5 Placebo Kp-va 10 40 thirty twenty 34 26 31 35 thirty D 29th 28 24 thirty twenty twenty 42 10 54 K-va 12 twenty 12 fifteen fifteen fifteen 5 twenty 10 Quality 35 44 23 23 twenty 13 48 37 average 25.3 30.7 18.0 22.5 19.0 19.3 15.0 27.5 26.0 33.7 Hematocrit Norm - from 35 to 45 0 After 1 XT After 2 XT After 3 XT 7 fourteen 21 7 fourteen 21 7 fourteen 21 Panagen Ev 40,2 38.1 40.8 37,4 34,4 39,4 36.6 33.6 34.6 B-ko 36 32.9 34.6 35,4 30.3 33,2 34.6 M-on 31.7 37,2 32,3 34.1 33.3 37.3 34.8 33.8 P-on 34.6 27 M-va 36.7 32 34,4 36.1 30.1 27.9 33.7 32,5 29.8 Md 31.1 29.5 35.6 36.5 34.2 32.9 30,2 31,2 30.9 B-r 28.6 26.2 26.8 25.7 26.9 26,4 27.8 27.5 24.1 Willow 47.7 35 32,5 33.7 29.9 29.8 34.9 32,7 31.8 thirty Eve 32 32,3 thirty 36.6 34.2 29.6 29.7 26.2 32.9 33,4 In 37,2 33.3 32,7 34.5 28.7 29th 35.9 33.6 33.8 36 Awa 39.2 41.2 29.5 35.3 23,4 22.6 G-ko 28,2 28.5 thirty 34.7 30.8 32,7 32,7 29.7 31.5 G-va 26.5 26,4 30.5 28.3 34.3 30.5 31.6 29.3 26.8 30.6 Cow 41.9 34.7 34.5 34.7 38.8 39.3 35,2 34.5 37.5 Peninsula 43.8 40 39.5 41.5 B-va 42,4 42.1 44.7 41.5 40,2 M-ko 44.3 41.2 38.5 38.5 40.8 average 37.9 34.9 32.0 * 35.0 32.4 * 31.4 * 34.8 31.2 * 31.1 * 34.0 Placebo Kp-va 37,2 37 34.8 37.5 32.1 35.7 35.6 31.3 35.6 D 34.1 33,4 29.1 36.1 34.9 36.5 36.6 33.5 34.9 K-va 35.3 32,5 35.1 36.7 32,7 30.7 36,2 31.6 35.1 Quality 37.1 32 30,4 34.3 25.6 31.3 33 29.8 28.9 28.1 Two 40,2 37.1 37.3 36 36,2 average 36.7 33.8 31.5 36.1 29.2 32.3 * 35,4 33,2 31.3 * 33.6

Estimation of the number of CD34 + / 45 + progenitor cells at control points during three XT courses.

Assessment of the number of CD34 + / 45 + hematopoiesis precursors was carried out on the 7th day after the administration of the cytostatic on the first XT and on the 21st day after each XT performed (Table 25).

Table 25 Percentage of CD34 + / 45 + cells in peripheral blood in patients receiving Panagen and placebo at various control points during 3 XT courses. 0 7 day after 1 XT 21 days after 1 XT 21 days after 2 XT 21 days after 3 XT Panagen Awa 1.20 0.05 0.15 0.05 0.09 B-r 0.02 0.10 0.14 0.09 0.08 Mr 1,60 0.60 0.09 0,03 0.04 Eve 0.09 0.16 0.26 0.22 0.25 Ev 0.02 0.24 0.04 0.11 Cow 1.90 0.05 0.19 0,03 0.15 M-va 0.05 0,03 0.30 0.22 0.31 M-on 0,03 0.08 0,07 Md 0.02 0.10 0.10 0.08 0.08 B-va 0.10 0.16 0.21 0.22 0.10 B-ko 0.08 0.02 0.17 0.28 Ch. Va 0.08 0.10 0.25 G-ko 0.02 0,03 0.25 0.70 0.30 G-va 0,03 0.08 0.15 0.20 0.10 P-on 0.04 0.20 Peninsula 0.06 0.16 0.23 0.31 In 0.14 0.12 0.26 0.14 0,07 Willow 0.38 0.01 0.66 0.50 0.20 M-ko 0.10 0.09 0.21 0.31 0.38 Placebo Two 0.15 0.20 0.33 0.28 0.20 D 0.12 0.01 0.20 0.31 Sh-va 0.08 0.04 0.55 Quality 0.05 0.08 0.19 0.45 0.08 K-va 0.05 0.05 0.20 0.30 0.43

As the analysis showed, the number of hematopoiesis precursors by day 21 of each subsequent XT was many times higher than the initial level both in the group taking the Panagen drug and in the placebo group (Fig. 31, table 26). This is due to the fact that therapies according to the schemes including the cytostatic CF, have an independent mobilizing effect on the hematopoietic precursors and are often used in combination with G-CSF to maximize the activation of proliferation and the release of neutrophilic cells into the blood.

Nevertheless, the analysis of the parameter on the 7th day after 1 XT showed a significant and significant difference with probability p _u <0.01 in the number of hematopoietic precursors between the Placebo and Panagen-responding groups and (Fig. 32, table 27) .

Table 26 The conditional content of CD34 + / 45 + cells in the peripheral blood in patients on the 21 control days after 3 courses of XT in relation to the initial level. After 1 XT After 2 XT After 3 XT Panagen responders M-va 638.3 468.1 659.6 In 185.7 100.0 50,0 Willow 173.7 131.6 52.6 M-on 266.7 233.3 Md 588.2 470.6 470.6 B-va 210.0 220.0 100.0 B-ko 212.5 350,0 B-r 666.7 428.6 381.0 Eve 288.9 244.4 277.8 Ev 1000,0 166.7 458.3 Ch. Va 125.0 312.5 G-ko 1250.0 3,500.0 1,500.0 G-va 454.5 606.1 303.0 M-ko 210.0 310.0 380.0 Peninsula 266.7 383.3 516.7 median 266.7 312.5 380.5 Panagen non-responding Awa 12.5 4.2 7.5 Mr 5.5 1.9 2,5 Cow 10.0 1,6 7.9 median 10.0 1.9 7.5 Placebo Two 220.0 186.7 133.3 D 166.7 258.3 Sh-va 687.5 Quality 380.0 900.0 160,0 K-va 400,0 600,0 860.0 median 380.0 429.2 160,0

Table 27 The conditional content of CD34 + / 45 + cells in peripheral blood in patients on the 7th control day after the first XT in relation to the initial level. Panagen responders B-r 476.2 B-va 160,0 Eve 177.8 Md 588.2 G-ko 165.0 G-va 230.3 P-on 500,0 median 230.3 Panagen non-responding Awa 4.2 Mr 37.5 Cow 2.6 M-va 68.1 B-ko 25.0 M-ko 90.0 In 85.7 Willow 3.2 median 31.3 Placebo Two 133.3 D 5.8 Quality 152.0 K-va 100.0 Sh-va 43.8 median 100.0

Evaluation of the functional activity of cells of innate anti-cancer immunity after three intensive XT courses

Using a cytotoxic test, the activity of innate anti-cancer immunity cells was assessed in the allogeneic system, which remained at the end of 3 XT (Fig. 33, table 28). This parameter testifies to the effect of the Panagen preparation on the state of the first echelon of the body's immunity after intensive cytostatic XT regimens for malignant neoplasms.

To assess the functional state of cytotoxic cells in the peripheral blood of patients in the study, a cytotoxic test was carried out using MCF-7 human breast adenocarcinoma cell culture as an appropriate target.

The cytotoxicity of the mononuclear fraction of human blood obtained by deposition of patients' peripheral blood on a ficoll-urograin gradient (density 1.077) was determined on the MCF-7 tumor cell line. Tumor cells were placed in a 96-well plate (5 × 10 ⁴ cells / well) and mononuclear cells were added in an amount of 5 × 10 ⁴ , 10 × 10 ⁴ or 25 × 10 ⁴ cells / well (1: 1, 1: 2 or 1 ratio :5). The cells were then incubated in RPMI-1640 medium with the addition of gentamicin sulfate (100 μg / ml) in an atmosphere of 5% CO ₂ at 37 ° C for 21 hours. After incubation, MTT solution was added to the wells to a final concentration of 0.5 mg / ml and cultured for 3 hours. Cells were centrifuged at 4000 rpm for 10 minutes (Eppendorf Centrifuge 5810 R). The medium was taken, the blue crystals of formazan precipitated were dissolved in 100 μl DMSO, the optical density was determined on a Multiskan RC instrument at a wavelength of 570 nm, subtracting the background at 620 nm. The MTT test results were processed using the Microsoft Excel 2002 program. Calculation of QI was carried out according to the standard formula:

QI (%) = [1- (OD _{e + m} -OP ₂ ) / OD _m ] * 100, where

OD _{e + m} - the value of optical density in the experimental series;

OP _e - the value of optical density in the wells with effectors;

OD _m - the value of optical density in the wells with targets.

Table 28 Cytotoxicity indices in patients at the starting point (0) and on day 21 after 3 XT in groups of patients receiving Panagen and placebo. 0 point Kor-va 52,5 Ev 18.5 Cr 33.8 3-va 74.8 Wow 26.5 Kp-va 58.0 Vd-on 37.0 T-on 68.0 median 44.8 Placebo - 21 days after 3 XT D 15.7 Quality 18.7 K-va 6.8 median 15.7 Panagen - 21 days after 3 XT B-ko 50,4 B-r 57.9 G-ko 78,4 G-va 41.6 Eve 38.3 Willow 42.1 Cow 63,2 M-va 49.9 M-on 32,0 median 49.9 Panagen non-responding In 6.9

Let us consider a generalized conclusion based on the results of a study of the leukostimulating effect of the drug based on the fragmented human DNA of Panagen during the sequential conduct of three XT using the FAC scheme for stage 2-4 breast cancer.

In the framework of studies of the drug Panagen as a leukostimulating agent, patterns were identified and results were obtained that characterize the effect of the drug on the hematopoietic sprouts of patients who are in the study during the increasing negative effect on hematopoiesis of consecutive XT courses.

The main objective of the study was to determine the effect of the drug on hematopoietic shoots in the growing progression of the negative effects of several consecutive XT. In this regard, two generalized parameters were necessary to evaluate the effect of the drug. The first is the dynamics of changes in the number of formed elements of the main hematopoiesis cells in peripheral blood at control points 1, 2 and 3 XT. The second is the number of shaped elements of a certain hematopoietic germ in peripheral blood at the last controlled point 21 days after 3 XT.

1. It was found that the drug differentially acts on different patients at different control points. In this regard, in each control point, 2 groups “responding” and “not responding” to the action of the drug were formed. The ratio of “respondents” to “non-responders” was on average 8.65 / 5.05 or 63.14% / 36.86%. Some parameters, for example, the absolute value of leukocytes and lymphocytes, at the control point 21 days after 3 XT had a significant difference between the Panagen and Placebo groups without dividing into responders and non-responders. The need for dividing into two subgroups was revealed during the study. The indicators of the “Panagen - not responding” group at this control point were so low that the emerging spread in the numerical values of the indicator did not make it possible to carry out comparisons using generally accepted methods for statistical estimation of the results. However, studies have shown that the Panagen - Not Responding group was not constant from one control point to another. Patients who did not respond at one control point could respond significantly to the effect of the drug at another. We attribute this phenomenon to “phase state” in the state of hematopoietic precursors of varying degrees of commitment.

In the state of HSC, there are biological phases when the cell perceives an inducing impulse or not. Such phases can be the presence of HSCs in a certain (G0) phase of the cell cycle, the state of exit from the hematopoietic niches and migration along the bloodstream associated with either the mobilizing effect of cytokines or the circadian rhythm of HSCs [39]. It is possible that the phases of susceptibility to the drug are connected with the effect of mobilization. It is known that HSCs emerging from hematopoietic niches due to the destruction of adhesive bonds when using G-CSF or CF mobilizing factors are not able to proliferate until they achieve bone marrow stroma and restore ectopic bonds with hematopoietic components niches. In the study of the cell cycle phase of circulating CD34 + cells, it turned out that the pool of mobilized G-CSF CD34 + cells contains less cells in the S phase and less cyclin activity is noted than in the pool of bone marrow CD34 + cells; in addition, they have lower metabolic activity [40–43]. Direct methods have shown that CD34 + cells mobilized by G-CSF are in the G0 / G1 phase of the cell cycle, while a large number of CD34 + cells were detected in the S + G2 / M phase in the bone marrow before the start of G-CSF [44] while the proliferative potential of mobilized HSCs is significantly lower than that of bone marrow cells. It can be assumed that the circulation and homing time of HSCs in patients is different. In this regard, there will be significant variations in the time of activation of HSC proliferation that returned to hematopoietic niches, which will affect the time of the appearance of the formed elements of the corresponding hematopoietic germ in peripheral blood. Since the control points are strictly fixed by the protocol, it is practically impossible to detect such a time shift in the HSC phases. And patients whose temporal activation parameters of the hematopoietic precursors are shifted in time will be in antiphase to patients “responding” to the effect of the drug.

Moreover, it is known that high-dose CF was the first mobilization protocol tested in clinical practice. When hemopoiesis was restored after myelosuppression in the peripheral blood, a more than 50-fold increase in the concentration of CFU-GM (granulocyte-macrophage colony forming unit), which is a precursor of monocytes and neutrophils, was recorded. The maximum titer of CD34 + HSC in peripheral blood was observed 5-7 days after cytostatic therapy. This cytostatic effect was the main difficulty in comparing and evaluating the effect of Panagen on the hematopoiesis of patients on the drug and placebo. Under the influence of CF, the whole system of hematopoietic precursors of the bone marrow comes to maximum tension, and it is extremely difficult to identify the effect of the hematopoietic action of another drug against this background, since it will be screened by the action of CF. This means that if under such conditions we find significant differences in the activation of hematopoiesis between patients on the drug and placebo, this means that the studied drug is a real activator of hematopoiesis in consecutive XT courses.

2. The critical point of cytostatic treatment is 14 days after exposure to cytostatics. At this control point, all indicators of the number of blood cells fall to critical values. It was found that the Panagen preparation helps to preserve the number of neutrophils in peripheral blood for 14 days from the use of cytostatics, which reduces the number of grade II neutropenia in this period of time by almost 2 times compared with the Placebo group (Fig. 34). There is also evidence that critical values of the neutrophil count at the control point 14 days after the use of cytostatics is not a prerequisite for the appointment of leukostimulants based on G-CSF. When using the Panagen preparation in the indicated mode, 1-3 days after the number of neutrophils drops to critical, their number will reach a non-critical value, and by the 21st day from the administration of cytostatics it will completely normalize. This in turn will allow the next XT course to be carried out on schedule, which is the key to high-quality therapy for malignant neoplasms.

3. It was shown that the effect of increasing the number of CD34 + / 45 + HSC in peripheral blood can be detected only on the 7th day of the first course of XT (Fig. 32). In other control points, no significant differences from the placebo group were found. As noted above, CF itself is a strong mobilizing factor. Significant differences found in the Panagen-responding group at the control point 7 days after 1 XT may be an indicator of the effective hematopoietic effect of Panagen.

Studies have shown that the drug Panagen activates CFU-GM granulocyte-macrophage and CFU-L lymphocytic hematopoiesis. Accordingly, when analyzing the number of formed elements in the blood of patients receiving Panagen and placebo, significant differences were found in the content of leukocytes in general, neutrophils, monocytes, lymphocytes and natural killers. No differences were found in evaluating the parameters characterizing the activation of CFU-E of erythrocyte and CFU-Meg megakaryocytic hematopoietic germs.

It is shown that the drug has a cumulative effect in the course of XT. While the “Placebo” group, all indicators to the last control point 21 days after the administration of cytostatics significantly decrease, the indicators in patients “responding” to the drug to the specified control point remain at a level significantly exceeding the most critical value found after 1 XT. The results suggest that the drug Panagen protects CFU-GM and CFU-L from inhibition by multiple sequential cytostatic treatments and at the same time induces the proliferation of these hematopoietic sprouts.

Separately, the effect of the drug in the direction of preserving and stimulating the proliferation of cells of innate anti-cancer immunity was analyzed (Fig. 33). An approach was used in which the non-specific cytotoxic activity of the mononuclear fraction of the patients included in the study was evaluated in relation to cells of the MCF-7 human adenocarcinoma cell culture. Studies have shown a protective and stimulating effect of the drug on this population of cells. The cytotoxic indices of patients taking the drug Panagen were significantly and significantly higher than in the placebo group.

Thus, the Panagen drug can be positioned in the list of leukostimulating drugs as a protector and stimulator of hematopoiesis of granulocyte-macrophage and lymphocytic hematopoietic sprouts against the background of intensive cytostatic chemotherapy regimens in cancer treatment regimens.

Consider the characteristics of the main indicators of the development of anti-cancer adaptive immunity in patients with breast cancer who received Panagen and placebo during the course of XT.

Consider the theoretical rationale for the activation of adaptive immunity in patients who participated in the study:

As part of the ongoing trials of a new drug based on Panagen human DNA, a parallel study was conducted related to the analysis of the development of adaptive anti-cancer immunity in patients participating in the protocol. This formulation of the question was associated with numerous experimental data obtained over the past 10 years, which unequivocally testified to the appearance of anticancer activity in the body of experimental animals using various strategies, the component of which was exogenous DNA of human origin. As evidenced by numerous and repeated experimental data, the main target of exogenous DNA in anticancer strategies was DC and the Th1 pathway induced by them, in which a pool of specific CD8 + T-cytotoxic lymphocytes is formed.

Along with the activation of the causal links in the development of anti-cancer adaptive immunity, strategies for treating cancer with high-dose cytostatic therapy are changing the number and functional state of regulatory T-lymphocytes, which, according to modern immunology, are responsible for suppressing the anti-cancer immune response.

In this regard, in the framework of ongoing studies of the peripheral blood of patients in the study, three areas of analysis were identified that could give either an unambiguously interpreted answer about the action of Panagen on the development of adaptive anti-cancer immunity, or indicate the further path of experimental work. The choice of the direction of the analyzes was also associated with the possibilities offered by the analysis of peripheral blood samples.

In this regard, the following parameters were evaluated, reflecting the activation of the development of anti-cancer adaptive immunity in the course of three XT conducted for stage II-IV breast cancer.

- The relative and absolute amounts of myeloid (CD11c + CD123-) and plasmacytoid (CD11c-CD123 +) DCs in the peripheral blood of patients were evaluated. Changes in the number of DCs, their subpopulation affiliation (but not the degree of maturity) were evaluated in the dynamics of therapy.

- The relative and absolute numbers of T-regulatory cells (CD4 + CD25 + FoxP3 + or CD4 + CD25 + CD127-) with suppressor activity were evaluated.

- The main attention was paid to assessing the dynamics of cytotoxic (CB8 + perforin +) T-lymphocytes as the main factors in developing anti-cancer adaptive immunity.

DC is a heterogeneous population of antigen-presenting cells of bone marrow origin. Morphologically, DCs are large cells (15–20 μm) of a round, oval or polygonal shape, an eccentrically located nucleus, and numerous branched processes of the membrane. DCs express a set of surface molecules characteristic of other antigen-presenting cells: receptors for components of the cell wall and nucleic acids of microorganisms, including receptors for complement components and toll-like receptors; MNSP molecules; co-stimulatory molecules CD40, B7 1/2 (CD80, CD86), B7-DC, B7-H1; intercellular adhesion molecules (ICAM-1). The main function of DC is the presentation of antigens to T cells. DKs also perform important immunoregulatory functions, such as controlling the differentiation of T-lymphocytes, regulating the activation and suppression of the immune response. An important feature of DC is the ability to capture various antigens from the environment through phagocytosis, pinocytosis and receptor-mediated endocytosis. Most DC is in tissues that come in contact with the external environment, for example, in the thickness of the epithelial layer of the intestinal mucosa, in the submucosa of the respiratory, gastrointestinal and urogenital tracts. DK absorb antigens, process and present on its surface in complex with MHC I or MHC class II. Only in this form, T cells are able to recognize the antigen and then activate and develop an immune response. Depending on the type of pathogen, DCs are able to direct the differentiation of naive T-helpers (Th0) towards T-helpers of type 1, T-helpers of 2 types, regulatory T-cells or T-helpers 17.

In humans, two subpopulations of DC are distinguished - myeloid and lymphoid DCs. Myeloid DCs are named so because they come from a common myeloid hematopoietic precursor. They are localized in various organs and tissues, where microorganisms and foreign proteins are captured by phage and pinocytosis, after which they express the antigenic determinant in combination with MHCII molecules. Then, DCs migrate to regional lymph nodes, where they stimulate the proliferation and differentiation of antigen-specific T-lymphocytes, thereby initiating and stimulating the immune response. Specific markers of myeloid blood DC are BDCA-1 (CD1c), BDCA-3 molecules. Myeloid DCs do not express markers of other cells of the immune system, such as CD14 (monocytes, macrophages and neutrophils), CD3 (T-lymphocytes), CD 19, CD20 (B-lymphocytes), CD56, CD57 (natural killer cells), CD66b (granulocytes) . Myeloid DCs are also characterized by the presence of the CD85k (ILT3) molecule. In response to stimulation by maturation inducers, myeloid DCs produce predominantly cytokines of the THY spectrum, including IL-6, IL-12, TNF-α and IFN-γ.

Lymphoid DCs are the main subpopulation of blood DC. The precursors of lymphoid DCs are a very small population of peripheral blood cells, characterized by the absence of the CD11 marker and the presence of the CD123 marker (IL3 receptor), which is called plasmacytoid DCs. Plasmacytoid DCs are capable of producing 200-1000 times more IFN-α than all other peripheral blood cells. Due to the production of the first type of interferons and IL-12, these cells have a modulating effect on the adaptive immune response, stimulate the maturation of myeloid DCs, as a result of which the T-cell response is polarized towards the Th1 variant [45]. Markers of plasmacytoid DCs also include BDCA-2, BDCA-4 molecules. The main population of lymphoid DCs express TRL-9, the ligands of which are bacterial DNA CpG oligonucleotides. Lymphoid DCs secrete IL-4 and IL-10, which switch the differentiation of zero T-helpers into type 2 T-helpers [46].

Myeloid DCs are able to activate CD8 + T cells. Lymphoid DCs cause the conversion of CD8 + T cells into suppressor (regulatory) cells. However, produced by the precursors of lymphoid plasmacytoid DC IFN of the first type, it promotes the maturation of myeloid DC, increases their ability to activate CD8 + T cells. This means that both myeloid and part of the lymphoid - plasmacytoid DCs participate in the formation of anti-cancer adaptive cellular immunity.

During the activation of the antitumor immune response, two pools of lymphocytes are formed. CD8 + T-lymphocytes, leading to the formation of a cellular and humoral response to progressive malignant tissue. As was said, myeloid DCs and part of lymphoid DCs (plasmacytoid DCs) participate in the formation of CD8 + populations. Lymphoid DCs take part in the formation of the humoral response, the main amount of which circulates in the peripheral blood [45].

During the formation of tumor tissue, DC precursors and immature CD4 + and CD8 + phenotypes migrate into it. Migration factor, apparently, are TNF-α or IL-1 cytokines. The migration process proceeds throughout the entire existence of the tumor [47]. Once in the tumor, immature DC precursors absorb various protein molecules, cell fragments from the extracellular space, as well as absorb proteins from the chaperones of the HSP family with antigenic peptide epitopes built into them. After the absorption of potential antigens in DC precursors, processes occur that lead to the presentation of antigenic epitopes together with histocompatibility molecules of classes I and II. It is important to note that the presentation takes place in both inactive and active DCs. However, in inactive DCs, this process has the character of a constant turnover of receptors from the outer surface of the cell inward and back. Upon activation, the departure of the MHCI and MHCII complexes from the cell surface is blocked, and the cell fully performs the antigen-presenting function [48]. The process of DC maturation induces two simultaneously occurring events - increased presentation of antigens and cell migration from the tumor to the regional lymph nodes. The migration process depends on the expression on their surface of the chemokine receptor CCR8, which responds to the chemokine gradient CCL1 (1-308), which regulate taxis in the lymph nodes [49]. Peptides generated by the processing of tumor-associated antigens are inserted primarily into MHCII molecules. Nevertheless, it has been shown that myeloid DCs are capable of presenting epitopes from protein molecules captured from the environment by class I histocompatibility molecules, which normally present epitopes of proteins synthesized in the cell itself. This phenomenon is called cross presentation. As a result of the cross-presentation mechanism, the presentation of peptides from tumor antigens is carried out both in the context of MHCI molecules and in combination with MHCII molecules. Simultaneously with the presentation of the antigen, co-stimulating B-7, ICOS molecules enter the cell surface and a set of cytokines is secreted. These events activate naive T cells, stimulating their development into functional CD4 + and CD8 + lymphocytes.

CD8 + lymphocytes are the main effectors of adaptive immunity. CD8 + T-cytotoxic lymphocytes and CD4 + Th1 cells are involved in the processes directly in the tumor growth site. Two mechanisms for the destruction of tumor cells by T-cytotoxic lymphocytes are suggested. This is a common effect of perforins, which are the main indicator of the cytotoxicity of CD8 + lymphocytes. Another mechanism is associated with the secretion of CD8 + by IFN-γ cells. It is believed that T-cytotoxic lymphocytes, secreting IFN-γ, enhance the cross-presentation of antigens in the endothelium of the tumor stroma and destroy tumor cells by activating the Fas receptor in them [50, 51]. Formed Th1 CD4 + helpers stimulate the formation of T-cytotoxic lymphocytes by activating DCs for their part. This process is carried out by IL-12 and IFN-γ secreted by Th1 helper cells. The second mechanism of the antitumor effect of Th1 cells is the induction of delayed type hypersensitivity, which consists in the stimulation of macrophages in tumor foci by secreted IFN-γ. Activated macrophages aggressively absorb tumor cells.

The population of Th2 T helper cells provides the formation of a humoral immune system response. Lymphoid (plasmacytoid) DCs can induce the formation of regulatory Th2 cells necessary for the formation of a humoral immune response. The initial stage of B-cell activation occurs at the border of the T- and B-cell zones of the secondary lymphoid organs. Antigenic stimulation induces their migration to T-cell zones. Here, B cells interact with antigen-specific Th2 helper cells. At the beginning, lymphoid (plasmacytoid) DCs activate the latter, which further recognize the antigen that they represent as B-cells in complex with MHC class II, and activate B-cells through the interaction of CD40L / CD40. Further differentiation of B cells into antibody-forming plasmocytes occurs in germination centers under the influence of specialized myeloid DCs.

Thus, the interaction of DC with cytotoxic lymphocytes has a complex bidirectional character. Their activation can carry out any DC. Due to the expression of IFN-1, IL-12, -15 and -18 DK, they increase the cytotoxicity of natural killers and the production of IFN-γ. Expression of CD40L or TNF-α induces maturation of lymphoid (plasmacytoid) DCs. IFN promotes the development of myeloid DCs and the expression of a Th1-differentiating signal. DK can both stimulate the T-cell response and cause immunological tolerance.

The presentation of antigens to cytotoxic T cells is carried out by MHC molecules of classes I and II. Sources of antigenic peptides represented by them are proteins synthesized in the DC itself, as well as exogenous proteins. Naive CD8 + T cells during the immune response can turn into non-polarized T-lymphocytes (CD45), secreting only IL-2; into effector cytotoxic T cells that produce the same cytokine spectrum as Th1 and Th2 cells. There is an additional subpopulation of cytolytic effector CD8 + T cells that secrete IFN-γ, expressing high levels of perforin and capable of immediate lysis of antigen-specific target cells.

And thus, there is a deep organic connection between the two populations of DC. Despite the fact that myeloid DCs containing a large amount of MNSP proteins on their surface are primarily responsible for the formation of a pool of CD8 + T-cytotoxic lymphocytes, plasmacytoid DCs are also directly related to the formation of the Th1 response, regulating the activation of myeloid DCs.

Consider the general ideological pattern of activation of an adaptive anti-cancer immune response when applying the claimed strategy.

1. XT involves the mandatory use of a combination of doxorubicin and CF (doxorubicin leads to the movement of calreticulin on the cytoplasmic membrane, which makes the tumor cell visible for attack by phagocytic cells, this is the “eat me - eat me” signal; CF leads to the destruction of the tumor cell and the formation of cell debris ; thus forming immunogenic cell debris).

2. At the same moment, CF differentially inhibits various populations of T-lymphocytes. So the inhibition of T-regulatory lymphocytes, which create anti-immune defense of the tumor, occurs almost completely. In this case, the suppressive function of T-regulatory lymphocytes is impaired. In this case, T-cytotoxic lymphocytes die only partially and are restored under the influence of the DNA preparation and DC activity. This creates a period of time when the tumor tissue becomes an unprotected cytokine gradient, protecting it from the effects of the immune system.

3. At the same time, the maturation is activated and the allostimulatory activity of the body DC in vivo is induced using the Panagen exogenous fragmented allogeneic DNA preparation. DCs mature and are loaded with tumor antigens that are now recognized by DCs. DCs activate the development of a T-cytotoxic specific anti-cancer response.

4. T-cytotoxic lymphocytes lyse everything related to their attack zone, including the remaining tumor cells.

5. T-memory is formed against this neoplastic material. Let us estimate the relative and absolute amount of myeloid (CD11C + CD123-) and plasmacytoid (CD11c-CD123 +) DCs in the peripheral blood of patients.

Since antigen-presenting cells, such as macrophages and DCs, originate from monocytes, stimulation with the Panagen preparation of a monocytic hematopoietic germ is a positive indicator of the therapy performed. In this regard, the assessment of the representation and ratio of myeloid (CD11 + CD123-) and plasmacytoid (CD11-CD123 +) DCs in the peripheral blood of patients in the study will characterize possible ways of activating the immune response. Analysis of the content of cytotoxic CD8 + perforin + lymphocytes in the peripheral blood will be a direct indicator of the development of anti-cancer adaptive immunity in patients participating in the clinical trials of the Panagen drug.

During the study, it was found that the regulation of the number of myeloid and plasmacytoid DCs differs both in the type of cells and in relation to different groups of patients. As in the case of the analysis of hematopoiesis stimulation with Panagen, it was found that some patients respond to the effect of the drug at this control point, and the other part does not respond. Although at a different control point the list of “responders” and “non-responders” could be different. In this regard, the following principle of analysis was chosen. Absolute indices were compared with respect to the zero point in the first and third XT separately. The indicators were compared with the control point 21 days after 1 XT, taken as 100%.

When analyzing the number of plasmacytoid DCs (CD11-CD123 +) in peripheral blood samples (table 29), it was found that out of a group of 14 people, 9 people respond to the drug's action in the aggregate after 1 and 3 XT, which is 64% (Fig. 35, table thirty). 36% of patients do not respond to the action of Panagen at the selected control points. During XT, in 30% of patients there is a significant increase in the number of plasmacytoid DCs in the peripheral blood to the last control point 21 days after 3 XT (Fig. 36, table 31). The remaining patients demonstrate a decrease or stabilization of this DC population in blood images in relation to the indicator after 1 XT.

Table 29 The percentage of CD11-CD123 + plasmacytoid DC in the peripheral blood of patients in the study. 0 - initial level 1 21 days after 1 XT 21 days after 3 XT Panagen Awa 0.42 0.57 0.16 B-ko 0.78 0.21 0.18 B-r 0.68 0.36 1.40 In 0.34 0.12 0.75 G-ko 0.88 0.54 0.52 Mr 1,56 0.85 0.74 G-va 0.13 0.12 0.80 Eve 0.96 0.61 0.49 Willow 0.34 0.30 0.64 Cow 2.00 5.26 0.84 M-va 0.29 0.90 0.10 Md 0.16 0.87 0.06 M-on 0.39 0.60 0.48 P-on 0.95 0.87 0.31 Placebo D 0.23 0.41 0.42 Quality 0.30 0.24 0.53 K-va 0.22 0.97 0.26

Table 30 The conditional content (%) of CD11-CD123 + plasmacytoid DCs in the peripheral blood of patients in the study was 21 days after 1 and 3 XT relative to the baseline. 21 days after 1 XT 21 days after 3 XT Panagen responders Awa 135.7 M-on 123.1 M-on 153.8 B-r 205.9 Cow 263.0 In 220.6 M-va 310.3 G-va 615.4 Md 543.8 Willow 188.2 median 263.0 median 205.9 Panagen non-responding B-ko 26.9 Awa 38.1 G-ko 61,4 B-ko 23.1 Mr 54.5 G-ko 59.1 Eve 63.5 Mr 47.4 P-on 91.6 Eve 51.0 B-r 52.9 P-on 32.6 In 35.3 Cow 42.0 G-va 92.3 M-va 34.5 Willow 88.2 Md 37.5 median 61,4 median 38.1 Placebo D 178.3 D 182.6 Quality 80.0 Quality 176.7 K-va 440.9 K-va 118.2 median 178.3 median 176.7

Table 31 The conditional content (%) of CD11-CD123 + plasmacytoid DCs in the peripheral blood of patients in the study was 21 days after 3 XT relative to the level after 1 XT. Panagen responders Panagen non-responding Placebo B-r 388.9 Awa 28.1 D 102,4 In 625.0 P-on 35.6 Quality 220.8 G-va 666.7 B-ko 85.7 K-va 26.8 Willow 213.3 G-ko 96.3 Mr 87.1 Eve 80.3 M-on 80.0 Cow 16,0 M-va 11.1 Md 6.9 median 506.9 median 57.8 median 102,4

The data obtained suggest that during therapy there is an increase in the population of circulating plasmacytoid DCs. In this case, a surge in the appearance of DC is associated with the condition of an individual patient. In our opinion, the effect of differences in the number of plasmacytoid DCs to 1 XT in some patients and to 3 XT in others is associated with individual characteristics. 63% of patients receiving Panagen demonstrate a response to the action of the drug by the totality of the control points 21 days after 1 and 3 XT.

Analysis of changes in the myeloid DC population (CD11 + CD123-) in the peripheral blood did not reveal patterns that would allow us to assess the possibility of activation of adaptive immunity through this population of antigen-presenting cells (Figs. 37, 38, tables 32-34).

Table 32 The percentage of CD11 + CD123-myeloid DC in the peripheral blood of patients in the study. 0 - initial level 21 days after 1 XT 21 days after 3 XT Panagen Awa 0.90 1.43 0.47 B-ko 0.93 0.51 0.18 B-r 0.68 0.87 0.99 In 0.59 0.37 1.05 G-ko 1.90 0.78 0.78 Mr 1,56 1,11 2.00 G-va 0.42 0.39 1.40 Eve 2.40 0.80 0.81 Willow 0.42 0.86 0.85 Cow 2.00 3.00 0.84 M-va 1.71 1.13 0.24 Md 0.18 1.20 0.15 M-on 0.88 0.86 0.75 P-on 1.05 1.32 0.38 Placebo D 0.32 0.32 1,02 Quality 0.80 0.35 1,60 K-va 0.97 1.46 0.30

Table 33 The conditional content (%) of CD11 + CD123-myeloid DC in the peripheral blood of patients in the study, 21 days after 1 and 3 XT relative to the initial level. 21 days after 1 XT 21 days after 3 XT Panagen responders Awa 158.9 In 178.0 B-r 127.9 B-r 145.6 Willow 204.8 Willow 202.4 Cow 150.0 Mr 128.2 Md 666.7 G-va 333.3 P-on 125.7 median 154.4 median 178.0 Panagen non-responding In 62.7 Awa 52,2 B-ko 54.8 Cow 42.0 G-ko 41.1 Md 83.3 Eve 33.3 P-on 36,2 M-va 66.1 B-ko 19,4 M-on 97.7 G-ko 41.1 Mr 71.2 Eve 33.8 G-va 92.9 M-va 14.0 M-on 85,2 median 64,4 median 41.1 Placebo D 100.0 D 318.8 Quality 43.8 Quality 200,0 K-va 150.5 K-va 30.9 median 100.0 median 200,0

Table 34 Conditional content (%) of CD11 + CD123-myeloid DC in the peripheral blood of patients in the study, 21 days after 3 XT relative to the level after 1 XT. Panagen responders Panagen non-responding Placebo B-r 113.8 Awa 32.9 D 318.8 In 283.8 B-ko 35.3 Quality 457.1 Mr 180.2 G-ko 100.0 K-va 20.5 G-va 359.0 Eve 101.3 Willow 98.8 Cow 28.0 M-va 21,2 M-on 87.2 Md 12.5 P-on 28.8 median 232.0 median 34.1 median 318.8

It is known that CF induces an increase in the number of DCs with the CD11 + phenotype on days 9–16 after injection [52]. DCs formed as a result of the action of CF activate the development of CD8 + T-cytotoxic lymphocytes. In this regard, it is rather problematic to evaluate the effect of the Panagen preparation by the appearance of certain populations of DC and cytotoxic cells in the peripheral blood under the conditions of the research protocol, since such an appearance will always be screened by the action of the CF itself. And, if under such conditions a reliable difference is found in the compared indicators, then the detected change objectively exists in the analyzed system.

The number of perforin-containing T-cytotoxic lymphocytes in the peripheral blood of patients in the study was estimated (table 35). The analysis showed that in 10 of 12 (83%) patients taking Panagen, the number of T-cytotoxic CD8 + perforin + in the first control point 21 days after 1 XT was significantly greater than in the placebo group (p _u <0, 05) (Fig. 39, table 36). If we consider the analyzed symptom in the totality of both control points, then in the vast majority of patients taking Panagen, the content of T-cytotoxic lymphocytes in peripheral blood is many times higher than the initial norm.

Table 35 The percentage of CD8 + perforin + cytotoxic lymphocytes in the peripheral blood of patients in the study. 0 - initial level 21 days after 1 XT 21 days after 3 XT Panagen Awa four 6 8 B-ko 6 8 B-r 10 fifteen 16 In 16 6 17 Ch. Va 9 5 G-ko 9 9 2 Mr 7 fifteen 6 G-va 3 9 13 Eve fourteen 17 7 Willow 17 eleven twenty Cow 2 four eleven M-va 17 21 10 Md 8 fifteen fifteen M-on fifteen 5 P-on 19 four Placebo D fourteen 9 Quality eighteen 8 19 K-va 9 eleven 10

Table 36 Conditional content (%) of CD8 + perforin + cytotoxic lymphocytes in the peripheral blood of patients in the study, 21 days after 1 and 3 XT relative to the initial level. 21 days after 1 XT 21 days after 3 XT Panagen responders Awa 150.0 Awa 210.0 B-r 147.0 B-r 160,0 G-va 300,0 G-va 420.0 Cow 200,0 Cow 525.0 G-ko 100.0 In 106.3 Mr 214.3 Md 178.6 Eve 121,4 Willow 117.6 M-va 123.5 B-ko 133.3 Md 178.6 median 148.5 median 178.6 Panagen non-responding In 39,4 G-ko 23.3 M-on 33.3 Mr 90.0 P-on 21.1 Eve 50,0 Ch. Va 55.6 M-va 58.8 Willow 61.8 median 39,4 median 54,4 Placebo D 64.3 D Quality 46.7 Quality 105.6 K-va 122,2 K-va 111.1 median 64.3 median 108.3

The data obtained indicate that in the body of patients taking Panagen during three XT for breast cancer, there have been changes that characterize the development of an adaptive immune response. Changes in the number of T-cytotoxic lymphocytes, apparently, is associated with the migration of lymphocytes to the tumor site.

Let us evaluate the relative and absolute number of regulatory T-lymphocytes in the peripheral blood of patients participating in the study of the leukostimulating effect of Panagen drug for stage II-IV breast cancer

As stated in the initial chapters of this study, CD4 + CD25 + Fox3 + T-regulatory lymphocytes occupy a special place in the induction of tumor resistance to counteracting the immune system. T-regulatory cells with the indicated phenotype play a central role in controlling the adaptive immune response and peripheral tolerance. A high concentration of these cells is detected in patients with breast cancer, colorectal cancer, lung cancer, pancreas and is an unfavorable prognosis. T-regulatory lymphocytes are formed from the same cells as T-helpers, under the influence of excessive concentrations of TGF-β, IL-10 and VEGF. The main mechanism of suppressive action of CD4 + CD25 + Fox3 + cells is associated with the secretion of suppressor cytokines TGF-β, IL-10, which inhibit the formation of IL-2 by effector cells. Also, T-regulatory lymphocytes can cause the elimination of T-lymphocytes through CD95 binding. At the same time, T-regulatory lymphocytes secrete cytokines inducing DC tolerance, while DCs do not express co-stimulating molecules and are not able to stimulate naive T-lymphocytes. Moreover, suppressors can induce the secretion of inhibitory cytokines by activated DCs themselves [53–57]. Thus, a sharp change in the number of suppressors CD4 + CD25 + Fox3 + may indicate significant changes in the relationship of the tumor and the immune system in the sense of its anticancer activity. The following is an analysis of the number of T-regulatory lymphocytes with the CD4 + CD25 + Fox3 + phenotype in the peripheral blood of patients in the Panagen study (Fig. 40, tables 37, 38).

Table 37 The percentage of CD4 + CD25 + Fox3 + T-regulatory lymphocytes in the peripheral blood of patients in the study. 0 - initial level 21 days after 1 XT 21 days after 3 XT Panagen Awa 10 9 eleven B-ko 13 9 9 B-r 6 7 5 In 5 9 8 G-ko 12 0.54 fourteen Mr eleven 2 10 G-va eleven 0.12 9 Eve 7 12 eighteen Willow four 7 12 Cow four 8 8 M-va 6 one 8 Md 10 12 6.2 M-on eleven fourteen 13 P-on 10 eleven 8 Placebo D 16 fourteen 10 Quality 7 10 fourteen K-va fourteen 12 5

Table 38 Conditional content (%) of CD4 + CD25 + Fox3 + T-regulatory lymphocytes in the peripheral blood of patients in the study, 21 days after 1 and 3 XT relative to the initial level. Panagen Panagen Change Placebo After 1 HT After 3 HT After 1 HT After 3 HT After 1 HT After 3 HT Awa 90.0 110.0 G-ko 4,5 116.7 D 87.5 62.5 B-ko 69.2 69.2 Mr 18.2 90.9 Quality 142.9 200,0 B-r 116.7 83.3 G-va 1,1 81.8 K-va 85.7 35.7 In 180.0 160,0 M-va 16.7 133.3 Eve 171.4 257.1 Willow 175.0 300,0 Cow 200,0 200,0 M-on 127.3 118.2 Md 120.0 62.0 P-on 110.0 80.0 median 123.6 114.1 median 10.6 103.8 median 87.5 62.5

As follows from the assessment, the following picture of the distribution of the number of T-regulatory lymphocytes in patients in the study is observed. Patients were divided into two groups. In the larger group, the number of T-regulatory lymphocytes in the first and second control points remains at the initial level, similarly to the placebo group. This one is a good prognosis. In 4 patients, which is 30% of the sample, the number of T-regulatory lymphocytes in the first control point 21 days after 1 XT fell 5-100 times. By the time of analysis at the second control point, 21 days after 3 XT, the number of suppressors had recovered to the initial initial level. In no group did not significantly increase T-regulatory lymphocytes. It can be assumed that this is primarily due to the use of CF in the treatment regimen, which, as shown repeatedly, selectively inhibits the development of T-regulatory lymphocytes and destroys the suppressor function of these cells [58-63]. Nevertheless, the result of the action of Panagen suggests that in some patients it is possible to repeatedly reduce the number of suppressors and thereby make the tumor accessible for attack by activated cytotoxic T cells.

Consider a generalized conclusion based on the results of a study of the influence of the applied strategy on the main indicators of the development of anti-cancer adaptive immunity in patients with breast cancer who received Panagen and placebo during XT courses.

Studies have shown that:

1. There are always two groups of patients, some of which respond to the action of Panagen, while others do not. The ratio of the number of patients “responding” and “not responding” in the total assessment of all the analyzed parameters was on average 50 to 50%.

2. Analysis of changes in the population of myeloid DCs (CD11 + CD123-) in the peripheral blood did not reveal patterns that would allow us to assess the possibility of activation of adaptive immunity through this population of antigen-presenting cells.

3. The data obtained suggest that during therapy there is an increase in the population of circulating plasmacytoid DCs (Fig. 36). In this case, a surge in the appearance of DC is associated with the condition of an individual patient. Individual characteristics are associated with the effect of differences in the number of plasmacytoid DCs to 1 XT in some patients and to 3 XT in others. 63% of patients receiving Panagen demonstrate a response to the action of the drug by the totality of the control points 21 days after 1 and 3 XT. Y 30% of patients taking Panagen, there is a significant increase in the number of plasmacytoid cells to 3 XT.

4. When analyzing the suppressor content of a larger group, the number of T-regulatory lymphocytes in the first and second control points remains at the initial level, similarly to the placebo group. In 4 patients, which is 30% of the sample, the number of T-regulatory lymphocytes in the first control point 21 days after 1 XT drops 5-100 times. By the time of analysis at the second control point, 21 days after 3 XT, the number of suppressors is restored to the initial initial level. In no group did not significantly increase the number of T-regulatory lymphocytes, which is a good prognosis.

5. In 10 out of 12 (83%) patients taking Panagen, the number of T-cytotoxic lymphocytes CD8 + perforin + at the first control point 21 days after 1 XT was significantly higher than in the placebo group (p _u <0.05) ( Fig. 39). If we consider the analyzed sign in the totality of both control points, then in all patients taking Panagen, the content of T-cytotoxic lymphocytes in the peripheral blood significantly exceeds the initial norm.

Studies have shown that in patients taking the drug Panagen, during three continuous XT induction of adaptive immunity occurred, which is a positive prognostic criterion for this group of patients. The data obtained can be evidence of the anti-cancer effect of the drug Panagen when used in combination with the cytostatics cyclophosphamide and doxorubicin in clinical use in the treatment of stage II-IV breast cancer.

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Claims (1)

A method of treating oncological diseases based on the use of cyclophosphamide and doxorubicin preparations and a preparation based on human exogenous DNA Panagen in the treatment schemes for stage II-IV breast cancer treatment schemes, characterized in that cytostatics are administered to the patient after a single chemotherapy cycle: cyclophosphamide ( 500 mg / m ² ) and doxorubicin (50 mg / m ² ) (AC scheme) or cyclophosphamide (500 mg / m ² ), doxorubicin (50 mg / m ² ) and fluorouracil (500 mg / m ² ) (FAC scheme) intravenously, as well as Panagen in the form of tablets, coated with an astroenteric coating at a dose of 30 mg / day with fractional uniform intake during the active period of the day, which is one tablet of the Panagen preparation six times a day every two hours with the start of administration from the third day after chemotherapy and continued administration of the drug for 17 days, and continued administration of the drug according to the specified scheme until the end of all courses of chemotherapy.

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