EA013806B1 - Use of erythropoetin for treating myeloblast leukosis - Google Patents

Abstract

The invention relates to medicine and provides the use of erythropoietin, in particular, erythropoietin-beta for the treatment of myeloblast leukemia at initial and developed stages and also to an optimum application of such use. The erythropoietin is an effective preparation expanding treatment abilities and enhancing the results of myoloblast leukemia therapy.

Description

State of the art

For 15 years, recombinant human erythropoietin has been widely used in oncohematology to prevent and treat anemia induced by intensive high-dose chemotherapy. The greatest effect of the use of erythropoietin was achieved in patients with non-myeloid tumors: non-Hodgkin's lymphoma, chronic lymphocytic leukemia, myeloma (1).

To date, numerous data have been accumulated indicating that, in addition to stimulating erythropoiesis, erythropoietin has a pronounced pleiotropic activity. In experimental studies, the effect of erythropoietin on tumor progression is studied. In particular, Μίί1е1шай с1 a1. (2) using models of mouse multiple myeloma MORS-315-1dA 1ashba and 5T33 MM-1dC, showed that daily administration of erythropoietin to mice with multiple myeloma for several weeks caused a complete regression of the tumor in 30-60% of individuals, increasing their lifespan and reducing mortality. Ka1x O. e1 a1. (3) Continuing experiments to study the effect of erythropoietin on the course of multiple mouse myeloma, it was established that not only CE8 (+) T-lymphocytes, but also activated B-lymphocytes are involved in antitumor reactions. The effect of erythropoietin on the acceleration of proliferation of splenocytes induced by ίη νίίτο lipopolysaccharide has been demonstrated.

In the experiments of ίη νίίτο and ίη νίνο, the possible mechanisms of the influence of erythropoietin on the sensitivity of malignant cells to chemo and radiation therapy are studied (4). Considering the issues of expanding the use of erythropoietin and its receptors, Ma1e8e K. eί a1. (5) note that they play an important role not only in the hematopoiesis system. C11ex / 1 R. eί a1. (6) analyze the mechanisms of action of erythropoietin that are not related to the stimulation of erythropoiesis, in particular, tissue protective and restorative effects. It has been established that erythropoietin reduces the manifestations of peripheral neuropathy and cardiotoxicity caused by chemotherapy. The use of erythropoietin as a cytoprotective agent for neurons and cells of the cardiovascular system is discussed. This applies to the appointment of erythropoietin in cerebral ischemia, myocardial infarction, chronic heart failure. Erythropoietin is a modulator of cellular processes such as stem cell development, cell integration, and angiogenesis. Erythropoietin inhibits the mechanism of apoptosis in trauma, including protecting the cell membrane and preventing inflammation. Studying non-hematological effects of erythropoietin and EPO receptors located on the cells of non-blood-forming tissues, Zeopd 8.V. her a1. (7) showed the possibility of restoring the growth of cell cultures PA \ U264.7 and PC12, from which fetal bovine serum was removed, by adding erythropoietin. The mechanism of cell growth restoration is associated with the expression of c-Po§ and e-1ip, as well as activation of the transcription factor AP-1. Important data were obtained by Nagoop Ζ.Α. her a1. (8) in an experiment consisting in subcutaneous implantation of a chamber from a semipermeable membrane in rats, where the process of replacing the fibrin in the chamber with a granulation tissue containing new blood vessels, macrophages and fibroblasts was studied, i.e. components of the reparative reaction. The introduction of recombinant erythropoietin into the fibrin matrix accelerated the formation of granulation tissue. To assess the role of endogenous erythropoietin in the healing process, soluble erythropoietin receptors and anti-EPO monoclonal antibodies were used to neutralize erythropoietin and cause dose-dependent inhibition of granulation tissue formation. It has been shown that the ability of erythropoietin to accelerate the wound healing process is associated with proangiogenesis occurring in granulation tissue. The expressed expression of EPO - receptors on macrophages - cells, which play a crucial role in wound repair, has been established. The ability to modulate the wound healing process by prescribing erythropoietin demonstrates a new direction in its use in the development of fibrin-induced wound healing.

A review article (9) provides evidence for the pleiotropic activity of erythropoietin. It has been proven that erythropoietin acts not only on erythroid cells, but also on myeloid, lymphocytes and megakaryocytes. This hormone is able to activate the phagocytic function of polymorphonuclear cells, thus modulating the inflammatory process. Erythropoietin receptors are found on endothelial and mesangial cells, on myocardiocytes, on smooth muscle cells and neurons. These findings stimulate studies of the hematologic effects of erythropoietin.

Currently, the unsolved problems of oncohematology include the limited use of erythropoietin in the treatment of myeloid tumors, in particular, myeloid leukemia.

Myeloid leukemia is a malignant disease that affects the hematopoietic myeloid growth and is accompanied by the accumulation of myeloblasts with the displacement of all other cells from the blood and bone marrow. The main modern method of treatment of myeloid leukemia is chemotherapy with the use of myelotropic cytostatic cytarabine, which can be considered as the closest analogue of the claimed invention. The disadvantages of this tool include its high toxicity, which leads to damage to vital organs and systems and the development of fatal complications.

Thus, the object of the invention is the search for a highly effective agent and the development of a scheme for its use for the treatment of myeloid leukemia. The problem is solved through the use of erythropoietin beta.

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The implementation of the invention

The mice of the мыши / 1Υ line were used as the object of study. The mice received standard laboratory food, water from car drinkers, and were kept in common cages at room temperature and natural daylight.

Mice of the ΆΚΚ line are short-lived, with an average lifespan of about 12 months and are characterized by a high frequency (80-95%) of the development of spontaneous leukemia aged 8 to 9 months. There are several subtypes of spontaneous leukemia that can be used to obtain transplantable leukemia.

In order to achieve the set objectives, a model of transplantable myeloid leukemia was chosen.

Method for the creation of transplantable leukemia

A mouse with obvious signs of spontaneous leukemia was decapitated. Under aseptic conditions, the spleen was removed from the abdominal cavity of the mouse, crushed with scissors, and rubbed with a glass rod on a nylon filter. A suspension of splenic cells was prepared on an isotonic sodium chloride solution to a final concentration of 0.01%.

To create transplantable leukemia, ΆΚΚ / 1Υ mice were selected at the age of 2-3 months. Intraperitoneally, each of them was injected with a prepared preliminary suspension of splenic cells of 0.5 ml.

Depending on the prevalence of the malignant process, transplantable leukemia is divided into three stages:

initial (moderate infiltration of certain organs by tumor cells);

detailed (severe infiltration by tumor cells of most organs); terminal (total metaplasia by malignant cells of all organs).

Features of the development of this model of transplantable leukemia are that the malignant process progresses much faster than anemia develops.

Evaluation of the effect of erythropoietin beta on the culture of hematopoietic progenitor cells isolated from mouse spleens

The aim of this study was to search for a dose of erythropoietin beta that effectively stimulates colony formation in a culture of murine hematopoietic progenitor cells.

A suspension of cells is prepared from the mouse spleen, from which the target cells are isolated at a concentration of 2-4 · 10 ⁴ and 1 ml of methylcellulosic medium Me1OiCi11 (81etCe11 TeiIboddez) is added. Cultivation is carried out for 14 days in a CO ₂ - incubator 8apuo - MSO. At the end of the cultivation, counting of colonies and clusters is performed using an inverted microscope LOMO Biolam P-1.

Conducted 10 experiments.

As a control, standard Me1oCi11 was used, containing 3 and 1 mt of erythropoietin.

Erythropoietin beta was added to experimental cups in the amount of 9 and t1, i.e. 3 times more than in the control and 28 and \ t1, i.e. 9 times more than in control. When the content of erythropoietin in the culture medium was 9 and t1, colony formation stimulation was not obtained. With the content of erythropoietin 28 and T1 in the medium, growth of macrophage colonies was noted. The results of the study are presented in table. one.

Table 1. The effect of erythropoietin beta on the colony forming ability of hematopoietic progenitor cells isolated from mouse spleen

The control 3 and \ t1 EPO (N = 3) Mixed colonies Erythroid colonies G ranulocyte colonies Macrophage colonies nineteen Erythropoietin 28 and \ t1 EPO (N = 7) one one 7 44

Thus, when conducting cultural studies, it was found that a dose of erythropoietin exceeding the normal level of erythropoietin by at least 9 times has a colony-stimulating effect. At the same time, growth of predominantly macrophage colonies was noted.

Study of the effect of erythropoietin beta on the course of myeloid transplantable leukemia in ΑΚΚ / 1Υ inbred mice

The experiments were performed on 56 mice (32 and 24?) Of the ΑΚΚ. / 1Υ line of generations P 210-212. The age of the males was 126 + 13.5 (n = 32) days. The body weight of males is 25.9 + 0.9 (n = 32) g. The age of the females was 117 + 5.5 (n = 24) days. The body weight of females is 23.0 + 0.55 (n = 24) g.

The mice were randomly divided into control and experimental groups with a restriction on

- 2 013806 randomization by age and body weight. All animals received the 94th passage of myeloid leukemia. On the same day, he began intraperitoneal administration of physiological saline (control group) of 0.5 ml and erythropoietin beta solution (experimental group) of 0.5 ml. The introduction was carried out daily, except for weekends. The dose of erythropoietin was 1000 IU / kg. A comparative assessment of life expectancy was carried out after 20 days.

20 days after inoculation of leukemia cells, the number of surviving mice of the experimental group was 15, 13 died. In the control group of live mice, 8 remained, and 19 died. In general, the average life expectancy of animals of the experimental group (n = 27) was 19.7 ± 0.306 days, in the control group (n = 25) - 18.7 ± 0.29 days (P <0.05).

Thus, erythropoietin beta provided an extension of life to mice of the ΑΚΚ / 1Υ line with transplantable myeloid leukemia.

In order to determine the most effective treatment regimen for myeloblastic inoculant leukemia in inbred ΛΚΡ / линии mice, the results of two experiments were compared. In the first erythropoietin beta was used for 13 days and in the second - for 7 days. There were no other differences between treatment regimens. Each experimental study included 4 options (physiological saline was administered in the first, erythropoietin in the 2nd, cytarabine in the 3rd, and erythropoietin with cytarabine in the 4th).

Statistical processing of material

All indicators are presented as mean ± one standard deviation of the mean (M ± t).

The research results were subjected to statistical processing using Student's 1-criterion, criteria for excluding pop-up values of the variant and nonparametric criterion and (1), as well as a mathematical apparatus for processing multivariate experiments (10.11).

The study of the effectiveness of treatment of myeloblastic transplantable leukemia in ΑΚΚ / ΊΥ inbred mice using erythropoietin beta for 13 days

The experiment was performed on 40 female mice of inbred lines of ΑΚΚ / ΤΥ generation E 212. The age of the mice was 124.4 ± 2.7 days. Body weight - 22.6 ± 0.73 g. Duration of the experiment - 20 days.

Mice were randomly divided into 4 variants with a restriction on randomization by age and body weight. All animals received the 99th passage of myeloid leukemia. On the same day, he began intraperitoneal administration of solutions according to the following scheme.

option - for four days a single injection of saline, then for five days a double injection of saline, then for four days a single injection of saline.

option - within four days the introduction of erythropoietin beta, then within five days the introduction of erythropoietin beta and saline, then within four days the introduction of erythropoietin beta.

option - for four days the introduction of saline, then for five days the introduction of cytarabine and saline, then for four days the introduction of saline.

option - within four days the introduction of erythropoietin beta, then within five days the introduction of erythropoietin beta and cytarabine, then within four days the introduction of erythropoietin beta.

The dose of erythropoietin was 1000 IU / kg. The dose of cytarabine was 0.003 mg per kg.

Were studied: the life expectancy of animals, weight loss during the study, the mass of organs after the experiment.

results

The life expectancy of mice, depending on the use of the studied drugs, is presented in table. 2.

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Table 2. Life expectancy of линии / ΊΥ mice with erythropoietin administered for 13 days

Statistic, indicators Date of Birth Date of death Age at the beginning of the experiment (in days) The life expectancy in the experiment (in days) Option 1 02/25/2007 08/06/2007 149 thirteen 02/25/2007 08/06/07 (3) 149 thirteen 03/15/2007 08/13/07 (3) 131 twenty* 03/15/2007 08/06/2007 132 thirteen 03/25/2007 08/07/2007 121 14 03/25/2007 08.08.2007 121 fifteen 03/26/2007 08/07/2007 117 14 03/26/2007 08/08/2007 117 fifteen 04/09/2007 08/12/2007 106 nineteen* 04/23/2007 08.08.2007 91 fifteen η 8 8 M 124,4 14 18.9 0.93 t 6.7 0.327 Option 2 02/25/2007 08/10/2007 149 17 02/25/2007 08.08.2007 149 fifteen 03/08/2007 08/09/2007 138 sixteen 03/19/2007 08/09/2007 127 sixteen 03/22/2007 08/06/07 (3) 124 thirteen 03/25/2007 08/09/2007 121 sixteen 03/26/2007 08/08/2007 120 fifteen 03/26/2007 08/12/2007 106 nineteen 04/09/2007 08/12/2007 92 nineteen 04/23/2007 08/10/2007 17 η 10 10 M 124.6 16.3 17.8 1,829 t 5,6 0.978 (P = 0.98) (P1- ₂ <0.01) Option 3 02/25/2007 08/11/2007 149 eighteen 02/25/2007 08/09/2007 149 sixteen 03/08/2007 08/10/2007 138 17

03/19/2007 08/08/2007 127 fifteen 03/20/2007 08/11/2007 126 eighteen 03/25/2007 08/09/2007 121 sixteen 03/26/2007 08/10/2007 120 17 03/26/2007 08/13/07 (3) 120 twenty 03/27/2007 08/06/07 (3) 119 thirteen 04/11/2007 08/10/2007 104 17 η 10 10 M 126.4 16.7 15.1 1,888 hl 4.8 0.597 (P = 0.8) (Ρ _{1. 3} <0,01ύ Option 4 03/08/2007 08/08/2007 138 fifteen 02/25/2007 08/09/2007 149 sixteen 03/08/2007 08/10/2007 152 17 03/19/2007 08/09/2007 126 sixteen 03/19/2007 08/09/2007 126 sixteen 04/25/2007 08/10/2007 90 17 03/25/2007 08/13/07 (3) 121 twenty 03/26/2007 08/09/2007 120 sixteen 03/27/2007 08/06/07 (3) 119 thirteen 04/11/2007 08/11/2007 104 eighteen η 10 10 M 126.2 16,4 22.1 1,838 hl 7 0.58 (P = 0.8) (P ₁ - ₄ <0.01)

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The results of measuring the mass of organs in mice (in grams) obtained during the experiment and at its completion are presented in table. 3.

Table 3. The masses of organs of mice of the ΛΚΚ / 1Υ line obtained in the experiment with the introduction of erythropoietin beta for 13 days

stat indicators M. spleen m. thymus m. hearts m liver m. kidney m. belly. l / y Option 1 0.3 0.155 0.142 2,55 0.339 0.164 0.35 0.198 0.107 * 2.2 0,207 0.104 0.415 0.137 0.133 2,4 0.249 0.25 0.384 0.145 0.138 1.54 * 0.138 * 0.15 0.283 0.136 0.125 2,4 0.346 0.335 0.372 0.175 0.138 2,57 0.297 0.117 0.405 0,111 0.135 2,5 0.297 0.188 0.457 0,101 0.128 2,32 0.313 0.113 0.385 0.1 0.114 2,55 0.275 0.237 0.33 0.176 0.123 2.47 0.3 0.131 η 10 10 nine nine nine 10 M 0.388 0.143 0.131 2.44 0.291 0.179 0,05534 0,0333 0.0089 0.123 0.0434 0,0715 t 0.0105 0.0105 0.003 0,041 0.0145 0,0236

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Option 2 0.533 0.11 0.135 3.2 0.314 0.287 0.6 0,053 0,132 3.15 0.286 0,203 0.609 0.14 0.135 2.75 0.289 0.149 0.697 0.135 0.128 3.05 0.3 0.244 0.326 * 0,076 0.13 1.5 * 0.275 0,085 0.696 0.105 0.158 * 3.17 0.302 0.152 0.579 0,094 0.123 2.65 0.138 * 0.225 0.469 0.0068 0.102 * 2.6 0.259 0.2 0.465 0,083 0.11 * 2.75 0.277 0.237 0.692 0.175 0.131 3.4 0.293 0.267 η nine 10 7 nine nine 10 M 0.593 0.104 0.131 2.97 0.288 0.205 0.0916 0,0374 0,042 0.286 0.0165 0,0614 ΠΊ 0,0305 0.0118 0.0016 0,095 0.0055 0.0194 Option 3 0385 0D85 η 1 ς? e 7 ^ ^< - / Ζ -ζ L 944 '-'l-' L 1Я7 0.265 0,067 0.125 2 0.291 0.173 0.33 0.139 0.139 2.2 0.28 0.215 0.382 0.135 0.118 2.27 0.29 0.145 0.347 0.103 0.131 2.15 0.332 0.2 0.365 0.121 0.139 2.59 0.33 0,086 0.319 0.08 0,132 2.19 0.353 0.335 0.415 0.137 0.133 2,4 0.249 0.25 0.052 * 0,037 0.109 0.97 * 0.226 not 0.299 0.121 0.137 1.95 0.277 0.122 η nine 10 10 nine 10 nine M 0.345 0,112 0,132 2.28 0.297 0.19 0,047 0.0423 0.012 0.262 0.0419 0,0734 t 0.0157 0.0134 0.0038 0,087 0.0132 0,0245 Option 4 0.487 0.122 0.134 2.95 0.295 0.184 0.472 0,088 0.136 2,3 0.291 0.202 0.444 0.127 0.122 2.1 0.251 0.262 0.549 0.127 0.141 2,57 0.329 0.155 0.41 0.109 0.137 1.95 0.272 0.105 0.61 0.103 0.15 3.15 0.33 0.339 0.411 0.15 0.117 2,55 0.244 0.35 0.62 0,091 0.141 2.77 0.297 0.221 0,031 * 0.044 * 0,096 * 0.77 * 0.218 not 0.514 0,112 0.155 3.4 0.323 0.169 η nine nine nine nine 10 d m 0.502 0.114 0.137 2.64 0.285 0.221 - 0,047 0.0195 0.012 0.48 0,0383 0.0825 t 0.0157 0.0065 0.004 0.16 0.0121 0.0275 P ( _{1. 2} ) <0.001 Ρ (ι- ₂ ) <0.05 P ( _{1. 2} ) <0.001 P (m) <0.01 Ρ (ι- ₄ ) <0.05 P ( ₂ - ₃ ) <0.001 P ( _{2. 3} ) <0.001 P ( ₂ -4) <0.05 P (s- ₄ ) <0.001

Mice were weighed before the start of the experiment and after completion (ροδί ИИННН). The results are presented in table. 4.

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Table 4. The results of a decrease in body weight of ΆΚΚ / 1Υ mice in an experiment with the introduction of erythropoietin beta for 13 days

stat indicators body initial m. body of roses! tog1aN5 Δ m.t. rsh.-m.t. ref. Option 1 34.5 32 -2.5 24 22.5 -1.5 26.8 21,4 -5.4 * 31.3 29.1 -2.2 21.9 22.2 0.3 23.9 21.3 -2.6 23.8 23.5 -0.2 19.9 twenty 0.1 ep η η and ζυ, ο ± /, / uh, ± 20.9 21.1 0.2 η nine M - 1.29 1.37 hl 0.456 Option 2 25.5 24.7 -0.8 24.5 25,2 0.5 22.3 22.7 0.4 25.8 24.8 -0.1 22 21.7 -0.3 25.8 25,2 -0.6 21.5 22.9 1.4 19.5 17,2 -2.3 20.7 18.1 -2.6 25.1 24.1 -one η 10 m -0.5 1.23 t 0.395 Option 3 eighteen 30.9 28.8 sixteen 23.7 22.4 17 23,4 25.1 fifteen 24.4 25.1 eighteen 23.9 21.8 sixteen 25.3 24.7 17 23 22.5 twenty 22.9 19.9 thirteen 20.7 20,2 17 21.3 21.3 η 10 M -0.77 1.41 t 0.445 Option 4

24.4 24.4 0 26.1 24.7 -1.4 twenty 20,2 0.2 27,2 26.5 -0.7 22.2 22.9 0.7 26.1 27.8 1.7 21.9 18.7 -3.2 * 25,2 24.7 -0.5 20.1 20.1 0 27,2 26.1 -1.1 η nine M - 0.122 0.948 w 0.316 Ρ (ΐ-4ΐ <0.05

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Thus, the life expectancy of LK./1U mice significantly increases in the 2nd, 3rd and 4th variants: with the introduction of erythropoietin, cytarabine and with their combined use.

The study of the mass of organs showed an increase in the mass of the spleen in the 2nd and 4th variants compared with the 1st variant, while the mass of the spleen in the 2nd variant was greater than in the 3rd and 4th, and in the 4th m more than in the 3rd.

The mass of the thymus was less in the 2nd variant than in the 1st and 4th.

The mass of the liver in the 2nd embodiment exceeds the mass of the liver of the 1st and 3rd variants.

A decrease in body weight was noted in all variants, but it was least pronounced in the 2nd and 4th variants.

The study of the effectiveness of treatment of myeloblastic inoculant leukemia in mice of the inbred line ΑΚΚ / линии when using erythropoietin beta for 7 days

The experiment was performed on 24 female mice of the AKK. / 1U inbred line of generation P 212. The age of the mice was 118.6 ± 3.76 days. Body weight 28.18 ± 1.48 g. The duration of the experiment is 20 days.

Mice were randomly divided into 4 variants with a restriction on randomization by age and body weight. All animals received the 101st passage of myeloid leukemia. On the same day, he began intraperitoneal administration of solutions according to the following scheme.

option - for four days, a single injection of saline, then for five days, a double injection of saline, then - until the end of the experiment, drug administration was not carried out.

option - for four days the introduction of erythropoietin beta, then for three days the introduction of erythropoietin beta and saline, then for two days of saline and then until the end of the experiment, the drug was not administered.

option - for four days, the introduction of saline, then for five days, the introduction of cytarabine and saline, then until the end of the experiment, the introduction of drugs was not carried out.

option - within four days the introduction of erythropoietin beta, then within five days the introduction of erythropoietin beta and cytarabine, then until the end of the experiment, the drug was not administered.

The dose of erythropoietin was 1000 IU / kg.

The dose of cytarabine was 0.003 mg / kg.

Were studied: the life expectancy of animals, weight loss during the study, the mass of organs after the experiment.

results

The life expectancy of mice, depending on the use of the studied drugs, is presented in table. 5.

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Table 5. Life expectancy of mice of the ΛΚΚ / 1Υ line with the introduction of erythropoietin for 7 days

Stat. indicators Date of Birth Date of death Age at the beginning of the experiment (in days) The life expectancy in the experiment (in days) Option 1 03/29/2007 09/04/2007 146 thirteen 04/05/2007 09/03/07 (3) 139 12 04/29/2007 09/03/2007 115 12 05/07/2007 09/06/07 (3) 108 fifteen 05/07/2007 09/04/07 (3) 108 thirteen 05/22/2007 09/05/2007 93 14 η 6 6 M 118.2 13,2 - 20.3 ± 1.17 t 8.3 ± 0.48 Option 2 04/02/2007 09/04/07 (3) 142 thirteen 04/03/2007 09/07/2007 141 sixteen 04/29/2007 09.09.2007 115 eighteen 01/07/2007 09.09.2007 228 eighteen 05/12/2007 09.09.2007 102 eighteen 05/12/2007 09.09.2007 102 eighteen η 5 5 M 120.1 17.6 twenty ± 0.89 w Option 3 8.9 (P-0.9) ± 0.4 04/02/2007 09/04/07 (3) 142 thirteen 04/05/2007 09.09.2007 139 eighteen 04/29/2007 09.09.2007 115 eighteen 05/03/2007 09.09.2007 111 eighteen 05/12/2007 09.09.2007 102 eighteen 05/12/2007 09.09.2007 102 eighteen η 6 5 M 118.5 eighteen 17.8 0 t Option 4 7.3 (P = 1) 0 04/03/2007 09/04/07 (3) 141 thirteen 04/05/2007 09.09.2007 139 eighteen 04/29/2007 09.09.2007 115 eighteen 04/29/2007 09/10/2007 115 nineteen 05/12/2007 09.09.2007 102 eighteen 05/21/2007 09.09.2007 93 eighteen η 6 5 M 117.5 18.2 19.3 ± 0.45 t 7.9 (P = 0.95) ± 0.2

The results of measuring the mass of organs in mice (in grams) obtained during the experiment and at its completion are presented in table. 6.

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Table 6. The masses of organs of mice of the line ΆΚΚ / 1Υ obtained in the experiment with the introduction of erythropoietin beta for 7 days

stat indicators M. spleen m. thymus m. heart m liver M. kidney m. belly. l / y Option! 0.25 0.128 0.188 2.82 0.433 0,096 0.25 0.141 0.166 2.2 0.38 0.196 0.27 0.038 * 0.119 2,5 0,038 0.185 0.442 0.17 0.158 3.45 0,303 0.259 0.136 0,101 0.136 1.22 0.295 0.04 0.366 0,132 0.124 2,35 0.271 0.194 η 6 5 6 6 6 M 0.286 0.134 0.148 2.42 0.344 • 0.106 0,0249 0,0268 0.74 0.0631 t 0.0432 0.0112 0,0109 0,301 0,0258

Option 2

0.331 0.168 0.137 1.29 0.271 0,071 0.784 0.253 0.179 * 4 0.41 0.182 0.44 0.114 0.125 2,5 0.296 0.218 0.448 0.138 0.14 3.05 0.361 0.25 0.626 0,091 0.142 2,8 0.29 0.234 0.605 0.13 0.125 2,8 0.296 0.273 η 6 6 5 6 6 M 0.539 0.149 0.134 2.74 0.321 0.1633 0,057 0.0082 0.878 0,0534 t 0,0666 0.0233 0.0037 0.398 0,0218 P p-21 <0.01 Option 3 0,063 * 0,063 0.151 1.32 0,327 Ί not 0.275 0.167 0.182 2.25 0.342 0.43 0.338 0,076 0.136 2.64 0.32 0.259 0.255 0.115 0.139 2.7 0.329 0.195 0.259 0.117 0.127 1.75 0.287 0.187 0.393 * 0.105 0.153 2.75 0.361 0.418 η 4 (6) 6 6 6 6 M 0.282 (0.283) 0.107 0.148 2.24 0.328 0.163 (0.112) 0,0365 0.0193 0.588 0,0246 t 0.066 (0.045) 0.0149 0.0079 0.239 0.0105 Rg2-zch <0.01 Option 4 0.239 0,094 0.14 1.37 0.395 0,055 0.491 0.108 0.185 * 2.69 0.415 0.287 0.494 0.145 0.143 2.59 0.314 0.214 0.299 0,067 0.133 1.8 0.31 0.174 0.367 0.205 0.135 1.9 0.319 0.362 0.554 0.134 0.142 2.93 0.356 0.15 η 6 6 5 6 6 M 0.407 0,126 0.139 2.21 0.352 0.1247 0,0479 0.0044 0.81 0,045 w 0,0509 0.0196 0.002 0.249 0.0184

Mice were weighed before the start of the experiment and after completion (ροδί ИИННН). The results are presented in table. 7.

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Table 7. The results of weight loss in mice of the AKELU line in an experiment with the introduction of erythropoietin beta for 7 days

stat indicators bm source M. body ροδί tog (a115 Amt.rt.m.t. Option 1 38.6 35.6 -3 42.5 38,4 -4.1 23.3 19.9 -3.4 33.8 32.1 -1.7 23 21.6 -1.4 23.1 21 -1.3 η 6 M -2.5 1.18 t 0.48 Option 2 33.1 29th '-4,1 * 42 40 -2 23,2 21.7 -1.5 23.3 23.9 '+0.6 22.3 22 -0.3 24 22.4 -1.6 η 5 M -0.98 1,08 t 0.48 Rya-2) ^< 0.05 Option 3 32 27.8 -4.2 36.7 33 -3.7 24 22 -2 21.3 19.3 -2 22 18.5 -3.5 22.3 23.7 '+0.7 η 6 M -2.45 1.7 t 0.73 Option 4 34.8 31.1 -3.7 38,2 34.9 s, s 24.2 22.6 -1.4 22.8 20.5 -2.3 21 20.6 -0.4 24.8 23 -1.8 η 6 M -2.15 1.22 t 0.5

Thus, there is a tendency to increase the life expectancy of mice of the AKELU line in the 2nd, 3rd and 4th variants: with the introduction of erythropoietin, cytarabine and with their combined use.

The study of organ mass showed an increase in spleen mass in the 2nd variant compared to the 1st

- 11 013806 and 3 options. Significant differences in the mass of organs between other options have not been established.

A decrease in body weight was noted in all cases, but it was least pronounced in the 2nd variant.

Comparative analysis of two treatment regimens for myeloblastic transplantable leukemia in ΑΚΚ / .1Υ inbred mice using erythropoietin beta

When assessing the differences in the change in body weight of mice during two experiments, it was found that with 13-day use of erythropoietin, the mass is least lost in the 2nd and 4th variants, and in the 4th variant, the body weight conservation is significantly higher than in 1st option (P <0.05). With 7-day use of erythropoietin, body weight is significantly preserved in the 2nd variant (P <0.05).

The life expectancy of mice, as well as the dynamics of spleen mass and liver mass, were analyzed using a two-factor experiment 2 ² [12, 10].

Each factor was investigated at two levels.

The first factor is the duration of the use of erythropoietin: the upper level is 13 days, the lower level is 7 days.

The second factor is the effect of cytarabine: the upper level - with the introduction of cytarabine, the lower level without cytarabine.

The table of the plan of the factor experiment is as follows:

Complete two-factor experiment 2 ²

Factors Response Function

Experience Number

.....

1M | I111I1m one III ··· | 11 ^ 2 +1 -one U. 111 ^ 1 I1 111I11 ΐΐΐΐΐιΜίι 4 +1 +1 at ₄

The plan consists of four experiments.

Experience No. 1 - both factors at the lower level: erythropoietin was used for 7 days, cytarabine was not administered.

Experience No. 2 - the first factor is at the upper level, the second factor is at the lower level: erythropoietin was used for 13 days, cytarabine was not administered.

Experience No. 3 - the first factor is at the lower level, the second factor is at the upper level: erythropoietin was used for 7 days, cytarabine was administered.

Experience No. 4 - both factors at the upper level: erythropoietin was used for 13 days, cytarabine was administered.

According to the results of the experiment, regression equations of the type y = b ₀ + b ₁ x ₁ + b ₂ x ₂ + b ₁₂ x ₁ x _{2 were} compiled, where y is the life expectancy of mice (in days);

x ₁ - erythropoietin factor;

x2 - cytarabine factor;

x ₁ x ₂ - the interaction of both factors (erythropoietin and cytarabine); B ₀ is the first term of the equation, the average value of the studied indicator of all four experiments of the factor experiment, b ₁ , b ₂ , b ₁₂ facing the signs of the factors are the coefficients indicating the strength of the influence of this factor. If the modulus of the coefficient | b | equal to or greater than the product of the error in determining the regression coefficient (8 _b ) by student criterion (1) - | b | > 8 _b -1, then this coefficient is considered reliable, and this factor has a statistically significant effect on the studied indicator. The + sign in front of the coefficient indicates that under the influence of this factor the studied indicator increases, the sign indicates that the studied indicator decreases under the influence of this factor.

The regression equation for estimating the life expectancy of mice in two experiments has the following form:

y = 16.8 - 1.05x ₁ + 0.25x ₂ - 0.05x ₁ x ₂ , where y is the life expectancy of mice in days;

x1 - erythropoietin factor;

x2 factor of cytarabine;

x ₁ x ₂ - the interaction of two factors; | B | = 0.3.

The coefficients facing the signs of factors x ₂ and x ₁ x ₂ are less than the critical value of 0.3, and before the factor x ₁ is greater than 0.3. Thus, y = 16.8 - 1.05 x ₁ . Consequently, under the influence of erythropoietin used for 7 days, the life expectancy of mice increases, and the use of erythropoietin for 13 days reduces the life expectancy. Cytarabine had no effect on

- 12 013806 life expectancy of animals. The combined use of erythropoietin and cytarabine also did not lead to an increase in life expectancy.

The regression equation for assessing changes in the spleen mass of mice in two experiments has the following form:

y = 0,533 + 0,022χι - 0,08h ₂ - 0,0065χιΧ _2, where - the change in mass of the spleen in grams;

χι - erythropoietin factor;

x ₂ - cytarabine factor; χιχ ₂ - the interaction of two factors; | B | = 0.046.

Thus, y = 0.533 - 0.08 x ₂ . Therefore, the mass of the spleen under the action of erythropoietin does not significantly change, and under the influence of cytarabine significantly decreases.

The regression equation for assessing changes in the liver mass of mice in two experiments has the following form:

y = 2.68 - 0.0225x! - 2.68x ₂ + 0.0175χ! Χ ₂ , where y is the change in liver mass in grams;

χ1 - erythropoietin factor;

x2-cytarabine factor;

χ1χ2 - the interaction of two factors; | B | = 0.21.

Thus, y = 2.68 - 2.68x2. Consequently, the mass of the liver under the action of erythropoietin does not significantly change, and under the influence of cytarabine significantly decreases.

Immunohistochemical study of the spleen in mice during experiments

When studying the mechanisms of the effect of erythropoietin on the course of myeloid transplantable leukemia, an immunohistochemical analysis of the spleen was carried out using polyclone 8-100 (ΚΤϋ) manufactured by Paco SuYutaiop. revealing interdigital dendritic cells in lymphoid tissue.

Evaluation of the results was carried out by the number of 8-100 positive (dendritic) cells in the spleen of mice in the field of view on the 13th day of the experiment.

It was found that regardless of the duration of erythropoietin administration, the content of 8-100 positive cells was significantly higher in the spleens of mice with inactive leukemia treated with erythropoietin and erythropoietin with cytarabine than in mice of the control group treated with saline and in mice treated with cytarabine.

Thus:

1. Erythropoietin beta effectively inhibits the development of transplantable myeloid leukemia of линии / 1Υ mice, which is manifested by an increase in life expectancy and the preservation of body weight of animals.

2. It has been established that providing an antitumor effect in the model of transplantable leukemia is associated with the use of high doses of erythropoietin beta, significantly exceeding erythropoiesis-stimulating doses.

3. An optimal scheme for the use of erythropoietin beta in the treatment of transplantable myeloid leukemia has been developed, which consists in the appointment of erythropoietin in the initial and advanced stages of leukemia. The use of erythropoietin in the terminal stage leads to the stimulation of the leukemia process.

4. In groups of mice with transplantable leukemia treated with erythropoietin beta, a high content of 8-100 positive (dendritic) cells was found in the spleen, which can be regarded as stimulation of stromal elements and as one of the possible mechanisms of the antitumor effect of erythropoietin beta.

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Claims (4)

CLAIM 1. The use of erythropoietin for the treatment of myeloid leukemia in the initial and advanced stages. 2. The use according to claim 1, characterized in that the erythropoietin is erythropoietin beta. 3. The use according to claim 1 or 2, characterized in that the doses of erythropoietin exceed erythropoiesis-stimulating doses by at least 3 times. 4. The use according to any one of claims 1 to 3, characterized in that erythropoietin is administered daily. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2