RU2699040C1 - Method for emergency prevention and treatment of acute radiation sickness (versions) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely to radiation medicine, and can be used for prevention and treatment of acute radiation sickness (ARS). First version of the method involves administering a liposomal preparation containing recombinant human alpha-protein (rhAFP) in amount of 0.1–1.0 mg/ml intravenously once after irradiation in a dose of up to 10 mg/kg of body weight in terms of rhAFP or same dose according to scheme: immediately after irradiation and 1, 3, 5, 7 days after irradiation. Second version of the method involves administering a liposomal preparation containing a recombinant human granulocyte colony-stimulating factor (rhGCSF) in amount of 0.004–0.06 mg/ml intravenously once immediately after irradiation in a dose of up to 0.2 mg/kg of body weight in terms of rhGCSF or in the same dose according to the scheme: immediately after irradiation and 1, 3, 5, 7 days after irradiation. In the third version, the acute prevention and treatment of acute radiation sickness is accompanied by administering a liposomal preparation containing rhAFP in amount of 0.1–1.0 mg/ml and rhGCSF in amount of 0.004–0.06 mg/ml. This preparation is administered intravenously once immediately after irradiation in dose of 10 mg/kg of body weight in terms of rhAFP and up to 0.2 mg/kg of body weight in terms of rhGCSF or in the same doses according to the scheme: immediately after irradiation and after 1, 3, 5, 7 days after irradiation.

EFFECT: use of the inventions enables the prevention and treatment of ARS by regulating growth, development and programmed death of hematopoietic stem cells, differentiation and activation of immune cells, stimulation of proliferation and maturation of granulocyte-containing blood leukocytes, as well as repair of radiation-damaged cell membranes.

3 cl, 4 tbl, 2 ex

Description

The group of inventions relates to medicines for therapeutic purposes, in particular, biotechnological materials, and can be used for the prevention and treatment of acute radiation syndrome.

The relevance of the problem can be explained as follows.

The use of nuclear weapons (including tests), accidents and accidents in industry, nuclear energy, medical and scientific institutions working with sources of ionizing radiation can lead to exposure of people.

The clinical course, severity and prognosis of radiation damage to the body are determined, on the one hand, by the absorbed dose of ionizing radiation, its distribution in space and time, and, on the other hand, by the radiosensitivity of tissues, organs and systems that are most important for the life of the body. Such tissues, organs and systems are usually called “critical”, since it is their damage that determines the predominant type of radiation reactions, the specifics and time of their occurrence, as well as the significance for the survival or death of the body at certain times after irradiation.

One of the clinical forms of radiation injuries is acute radiation syndrome (OLS), which develops after short-term (from seconds to 3 days) exposure to ionizing radiation in doses exceeding the average per body in 1 Gy. According to ICD 10, OLS refers to Section XIX “Injuries, Poisoning and Some Other Consequences of Exposure to External Causes”, is included in S00-T98 “Other and Unspecified Effects of Exposure to External Causes”: T66 “Unspecified Effects of Radiation”.

In the dose range of up to 10 Gy, the hematopoietic system is a critical system. In this case, a bone marrow form of OLS develops, a mandatory part of which is the clinically recognizable radiation depression of hematopoiesis (acute radiation bone marrow syndrome), hematopoietic tissue hypoplasia, associated pancytopenia and its complications - cytopenic infections, bleeding and anemia. The survival of hematopoietic stem cells determines the severity and outcome of the bone marrow form of OLS, in addition, transplantation of these cells is one of the most effective tools for the treatment of acute radiation syndrome [Eric J. Hall, Amato J. Giaccia Radiobiology for the Radiologist // Lippincott Williams & Wilkins, 2012 - 576 p. TM Fliedner, NJ Chao, JL Bader, A Boettger, C Case, J Chute, DL Confer, A Ganser, NC Gorin, P Gourmelon, DH Graessle, R Krawisz, V Meineke, D Niederwieser, M Port, R Powles, In Sirohi , DM Weinstock, A Wiley, and CN Coleman Stem Cells, Multi-organ Failure in Radiation Emergency Medical Preparedness: A US / European Consultation Workshop // Stem Cells. 2009 May; 27 (5): 1205-1211.]. The lethal dose of LD50 / 30 with total external gamma radiation shows that the survival of animals is determined, inter alia, by the number and radiosensitivity of hematopoietic stem cells [Van Bekkum D.W. Radiation sensitivity of hematopoietic stem cell. // Radiat Res. 1991 Oct., 128 (1 pc.): S4-8.]. This dependence is most clearly defined for mice in which it is possible to assess the survival of hematopoietic stem cells using colony forming units in the spleen (CFUc) [van Bekkum D.W. Radiation sensitivity of hematopoietic stem cell. // Radiat Res. 1991 Oct., 128 (1 pc.): S4-8.].

Therefore, the study of the survival of hematopoietic stem cells is one of the informative tools for assessing the effectiveness of the radioprotective properties of drugs [Pereverzev AK Hematopoietic colony-forming cells and physical stress factors. // L .: Nauka, 1986. 172 p. Horizons P.D., Belousova O.I., Fedotova M.I. Stress and blood system. M .: Medicine, 1983. 240 p. Konoplyannikov A.G. Stem cell radiobiology. M .: Energoatomizdat, 1984. 120 p.]

It is known from the prior art that liposomes are used as universal means of delivering drugs to pathological foci - tumors, inflamed tissues, (see. Appendix Baryshnikova A.Yu. “Nanostructured liposome systems as a means of delivery of antitumor drugs” in the journal “RAMS” , 2012, No. 3).

So, in this article, the properties of liposomal preparations are sufficiently fully characterized. In particular, the following is described.

“Liposomes are hollow particles whose contents are limited by the lipid membrane. They belong to the family of vesicular structures formed by amphiphilic molecules. The universal properties of the liposomal dosage form explain its wide possibilities, especially in cancer chemotherapy. The main purpose of using liposomes as drug carriers is the selective accumulation of active substances in pathological foci. Simple liposomes are composed only of phospholipids (neutral and / or negatively charged) and / or cholesterol.

Introduced in various ways in vivo, they circulate in the bloodstream for a fairly short period of time and quickly accumulate in the mononuclear phagocytic system, known as the reticuloendothelial system (RES). The main places of accumulation of liposomes are the liver and spleen. A large number of phagocytes and abundant blood supply are the main reasons for the capture of vesicles mainly by the liver and spleen. Due to this property, liposomes can be used for delivery to macrophages of immunomodulators, cytotoxic and antimicrobial compounds by passive targeting "

As the closest analogue to the one claimed in terms of the use of liposomal preparations, the known method of treating local radiation injuries of the skin using liposomal systems was described, described in clause RF No. 2642957, in which, along with the method of treating skin radiation burns, the liposomal preparations used are described in detail.

The formula of the known patent is as follows.

1. Liposome for the treatment of local radiation lesions of the skin, containing human alpha-fetoprotein.

2. The liposome according to claim 1, characterized in that said human alpha-fetoprotein is a recombinant human alpha-fetoprotein.

3. The liposome according to claim 1, characterized in that said liposome contains from 0.1 mg / ml to 1.0 mg / ml of human alpha-fetoprotein.

4. The liposome according to claim 1, characterized in that said liposome further comprises a human granulocyte colony stimulating factor.

5. The liposome according to claim 4, characterized in that said human granulocyte colony-stimulating factor is a recombinant human granulocyte colony-stimulating factor.

6. The liposome according to claim 4, characterized in that said liposome contains from 0.004 mg / ml to 0.06 mg / ml of granulocyte colony stimulating human factor.

7. The use of liposomes according to any one of paragraphs. 1-6 for the treatment of local radiation lesions of the skin.

8. A pharmaceutical composition for treating local radiation injuries of the skin, comprising an effective amount of liposomes according to any one of 1-6.

9. A drug for the treatment of local radiation lesions of the skin, containing the pharmaceutical composition according to p. 8.

10. The drug according to p. 9, characterized in that the tool is a means of transdermal action.

11. The drug according to claim 9 in the form of a plate, patch, film, cream, ointment or spray.

12. A method of treating and / or preventing local radiation injuries of the skin, comprising the step of applying a pharmaceutical composition according to claim 8 or a drug according to claim 9 onto an affected or affected area of the skin.

Summarizing the above on a well-known patent, we can formulate a known method, used below as a prototype, as follows.

A method for the treatment and / or prevention of local radiation injuries of the skin, including the use of liposomes with recombinant human alpha protein (rhAFP) in an amount from 0.1 mg / ml to 1.0 mg / ml or / and recombinant human granulocyte colony stimulating factor (rhGKSF) in an amount of from 0.004 mg / ml to 0.06 mg / ml and its direct use in the affected organ by application it on the affected area of the skin in the form of a plate, patch, film, cream, ointment or spray.

A disadvantage of the known method of treatment using these drugs is that they are intended and can only be used to treat radiation damage to the skin.

The objective of the proposed technical solution is to ensure the possibility of prevention and treatment using the above drugs of acute radiation syndrome

The problem is solved in that:

- in the first embodiment, in the method of emergency prevention and treatment of acute radiation syndrome, including the use of a liposomal preparation containing recombinant human alpha protein (rhAFP) in an amount of 0.1-1.0 mg / ml, ACCORDING TO THE INVENTION, the liposomal preparation is administered intravenously once immediately after irradiation in a dose of up to 10 mg / kg body weight in terms of rfAPP or in the same dose according to the scheme: immediately after irradiation and 1, 3, 5, 7 days after irradiation.

- in the second embodiment, in the method of emergency prophylaxis and treatment of acute radiation syndrome, including the use of a liposomal preparation containing recombinant human granulocyte colony stimulating factor (rhGCSF) in an amount of 0.004-0.06 mg / ml, according to the INVENTION, the liposomal preparation is administered intravenously once immediately after irradiation in a dose of up to 0.2 mg / kg body weight in terms of rhGKSF or in the same dose according to the scheme: immediately after irradiation and 1, 3, 5, 7 days after irradiation.

- in the third embodiment, in the method of emergency prevention and treatment of acute radiation syndrome, including the use of a liposomal preparation containing recombinant human alpha protein (rhAFP) in an amount of 0.1-1.0 mg / ml and recombinant human granulocyte colony stimulating factor (rhGKSF) in an amount of 0.004-0.06 mg / ml, ACCORDING TO THE INVENTION, a liposome preparation is administered intravenously once immediately after irradiation at a dose of up to 10 mg / kg body weight in terms of rfAPP and up to 0.2 mg / kg body weight in terms of rhGCSF or in those same scheme doses: immediately after irradiation and after 1, 3, 5, 7 days after irradiation.

In the first embodiment, the use for the treatment of a combined liposome preparation containing recombinant human alpha protein at its maximum dose (up to 10.0 mg / kg body weight) is able to regulate the growth, development and programmed death of hematopoietic stem cells, as well as regulate differentiation and activation immune cells, allows in combination with the use of phospholipid liposomes with high permeability when administered intravenously with the above scheme is effectively ive it to treat emergency and prevention of acute radiation syndrome. In addition, the phospholipids from which liposomes are formed are the material for the repair of radiation-damaged cell membranes, which leads to increased survival of hematopoietic stem cells after radiation exposure and also increases the efficiency in combination with the fact that liposomes are a means of delivery of a regulatory protein (rhAPP) into the cell by a mechanism independent of the receptors for this protein.

In the second embodiment, the use of a recombinant human granulocyte colony-stimulating factor (rhGCSF) in its maximum possible amount (up to 0.2 mg / kg body weight), which stimulates the proliferation, maturation and function of granulocyte-containing blood leukocytes, in combination with phospholipid liposomes, having a high penetrating ability into cells and being a material for the repair of radiation-damaged cell membranes, which leads to increased cell survival after radiation exposure, poses when introducing it according to the above scheme intravenously in the above doses, significantly increase the mass of the spleen and the number of colony forming units in the spleen by increasing the survival and proliferative activity of hematopoietic cells, providing effective treatment and prevention of acute radiation syndrome.

In the third embodiment, the use for the treatment of a combined liposome preparation containing recombinant human alpha protein at its maximum dose (up to 10.0 mg / kg body weight), capable of regulating the growth, development and programmed death of hematopoietic stem cells, as well as regulating differentiation and activation of immunocompetent cells, together with a recombinant human granulocyte colony stimulating factor (rhGKSF) in its maximum possible amount (up to 0.2 mg / kg body weight) stimulating the proliferation, maturation and function of granulocyte cells in the bone marrow when phospholipid liposomes are used for delivery to stem hematopoietic cells, which have a high penetrating ability into the cells and serve as a material for the repair of radiation-damaged cell membranes, which leads to an increase in their survival after radiation effects, while liposomes are a means of delivering regulatory proteins (rhAFP and rhGKSF) into the cell, according to a mechanism independent of the receptors for these proteins , and that two proteins (rhAFP and rhGKSF) enhance the stimulating effect of each other on hematopoietic stem cells, when a combined preparation is administered intravenously according to the above scheme, it can be used even more effectively for emergency prevention and treatment of acute radiation syndrome.

The technical result in all three variants is the provision of the possibility of effective treatment and prevention of acute radiation syndrome.

Compared with the prototype, the claimed option has novelty in all three of its variants, differing in such essentials, which together provide the desired result, essential features, such as:

- in the first option - the introduction of a liposomal preparation containing only recombinant human alpha protein, intravenously once immediately after irradiation at a dose of up to 10 mg / kg body weight in terms of rfAP or in the same dose according to the scheme: immediately after irradiation and after 1, 3, 5, 7 days after irradiation;

- in the second option - the introduction of a liposomal preparation containing only a recombinant granulocyte colony stimulating factor in humans, intravenously once immediately after irradiation in a dose of up to 0.2 mg / kg body weight in terms of rhGCSF or in the same dose according to the scheme: immediately after irradiation and through 1, 3, 5, 7 days after irradiation;

- in the third embodiment, the introduction of a liposomal preparation containing both a recombinant human alpha protein and a recombinant human granulocyte colony stimulating factor, intravenously once after irradiation at a dose of up to 10 mg / kg body weight in terms of rfAP and up to 0.2 mg / kg body weight body in terms of rhGKSF or in the same doses according to the scheme: immediately after irradiation and 1, 3, 5, 7 days after irradiation.

Although from the clause of the Russian Federation No. 2642957, such drugs as a liposome with recombinant human alpha protein (rhAFP = 0.1 mg / ml to 1.0 mg / ml) are known per se, a liposome with recombinant human granulocyte colony stimulating factor (rhGCSF) = from 0.004 mg / ml to 0.06 mg / ml), as well as a liposome containing both rhAPP and rhGKSF in the above amounts, however, they are used to treat only radiation injuries of the skin and are intended for external use. The use of liposomal drugs with the indicated drugs encapsulated in liposomes by intravenous administration of them in the above doses and according to the above scheme for the treatment of acute radiation syndrome is not known to the applicant, therefore, he considers that the claimed method in all its three variants meets the criterion of “inventive step” .

The inventive method in all three of its variants can be used for the treatment and emergency prevention of acute radiation syndrome and therefore meets the criterion of "industrial applicability".

The inventive method is as follows.

In the first embodiment, for emergency prophylaxis and treatment of acute radiation syndrome, a liposomal preparation containing recombinant human alpha protein (rfAPP) in an amount of 0.1-1.0 mg / ml is used. This drug is administered intravenously once immediately after irradiation in a dose of up to 10 mg / kg body weight in terms of rfAPP or in the same dose according to the scheme: immediately after irradiation and 1, 3, 5, 7 days after irradiation.

In the second embodiment, for emergency prophylaxis and treatment of acute radiation syndrome, a liposomal preparation containing recombinant human granulocyte colony stimulating factor (rh-GCSF) in an amount of 0.004-0.06 mg / ml is used. This drug is administered intravenously once immediately after irradiation in a dose of up to 0.2 mg / kg body weight in terms of rhGKSF or in the same dose according to the scheme: immediately after irradiation and 1, 3, 5, 7 days after irradiation.

In the third embodiment, for emergency prevention and treatment of acute radiation syndrome, a liposome preparation is used containing recombinant human alpha protein (rhAFP) in an amount of 0.1-1.0 mg / ml and recombinant human granulocyte colony stimulating factor (rhGKSF) in an amount of 0.004-0 , 06 mg / ml. This drug is administered intravenously once immediately after irradiation at a dose of up to 10 mg / kg body weight in terms of rhAFP and up to 0.2 mg / kg body weight in terms of rhGKSF or in the same doses according to the scheme: immediately after irradiation and after 1, 3, 5, 7 days after irradiation.

In practice, the inventive method in different versions of its implementation is as follows.

To implement the method, the following materials and methods were used.

As a biological object in the experiments, male CD1 and C57b1 / 6 mice were used, aged 3-4 months, weighing 24-26 g, obtained from the nursery NPP Nursery for laboratory animals FIBH RAS (Pushchino, Moscow region. )

Test Subject Characterization

In experiments on mice, drugs obtained at the Institute of Engineering Immunology of the FMBA of Russia were tested (drug passports are given in Appendix A of the Report).

1. Empty liposomes contain 10 mg / ml phospholipids (Appendix A. Preparation A0).

2. Liposomes containing 1 mg / ml rhAFP and 0.02 mg / ml rhGKSF, 10 mg / ml phospholipids (Appendix A. Preparation A4).

3. Liposomes containing 1 mg / ml rhAFP, 10 mg / ml phospholipids (Appendix A. Preparation A2).

4. Liposomes containing 0.02 mg / ml rh-GCSF, 10 mg / ml phospholipids (Appendix A. Preparation A3).

5. A solution of rhAFP protein containing 1 mg / ml rhAFP (Appendix A. Preparation A6).

6. A solution of rhGCSF protein containing 0.02 mg / ml rhGCSF (Appendix A. Preparation A7).

To assess the effect of individual components (liposomes, rhAFP, rhGCSF) and their combinations on the state of bone marrow stem cells, it was performed using the endotest method [67]. The study was performed using male CD1 stock mice and C57b1 / 6 mice.

The experimental design of the evaluation of the influence of the studied drugs on the state of HSC using mice runoff CD1 are shown below in table 1.

The experimental design for assessing the effect of the studied drugs on the state of HSC using mice of the C57b1 / 6 line is shown in Table 2 below.

Below are examples of the results of the experiment of a single administration of drugs after gamma radiation (example 1) and multiple (course) use of drugs after gamma radiation (example 2) ..

EXAMPLE 1. A single injection of drugs after gamma radiation. The experimental results showed that on the 9th day after the irradiation of mice, the number of endocolonies in the spleen was 3.4 ± 0.69 CFU / s; spleen mass - 31.87 ± 1.87 mg. Table 3 below shows the endotest indices in C57Bl / 6 mice with a single injection after irradiation

Note: CFUs are colony forming units in the spleen; YSC KM - nucleated cells in the bone marrow.

The administration of empty liposomes to irradiated animals (table 2, group 2) led to a significant increase in the number of CFUs in mice by 150% relative to the irradiated control and an increase in spleen mass by 27%, but these changes did not reach statistical significance (t _st = 2.19 ; p = 0.06).

The most pronounced effect was recorded in the experimental group, where animals received liposomes containing rhAFP and rhGKSF (table 2, group 5). In this case, the number of CFUs exceeded that of the irradiated control by 247%; the number of YSC in the bone marrow - by 123%; spleen mass - by 45%.

The introduction of mice liposomes containing rhAPP (table 2, group 3) also led to a significant increase relative to the irradiated control number of CFUs (by 190%), spleen mass (by 60%).

The administration to mice after irradiation of liposomes containing rhGCSF (table 2, group 4), an rhGCSF protein suspension and rhAFP protein suspension did not lead to a significant change in the studied parameters relative to the irradiated control group.

EXAMPLE 2. Repeated (course) use of drugs after gamma radiation.

A second series of experiments to evaluate the effect of drugs on the hematopoietic stem cell (HSC) was performed using CD1 mice. In this case, the mice were irradiated at a dose of 4 Gy and intravenous administration of preparations was carried out 5 times according to the scheme shown in table 1.

The results of the endotest indices in mice CD1 runoff with the introduction of drugs after 5 minutes and further on 1, 3, 5, 7 days after irradiation, are presented below in table 4. In the irradiated control group (table 1, group 1), the number of CFUs is 15.2 ± 3.59 CFUs / sel .; spleen mass - 65.94 ± 9.47 mg.

Note: CFUs are colony forming units in the spleen; YSC KM - nucleated cells in the bone marrow; t is the value of the Student coefficient when compared with indicators in the group of irradiated control + saline; p is the level of statistical significance.

When administered five times, the most pronounced effects were recorded in mice treated with liposomal preparations containing rhAPP. So in the group where mice were injected with rhAFP-containing liposomes (table 1, group 3), the number of CFUs was significantly increased by 171%, and the spleen mass was 129% relative to the irradiated control. In the group where the animals were injected with liposomes containing rhAFP and rhGKSF (table 1, group 5), the number of CFUs was significantly increased by 196%, and the weight of the spleen - by 199%. It should be noted that part of the animals of this group observed confluent growth of colonies. When animals were injected with a suspension of rhAFP protein (table 1, group 6), a significant increase in the number of CFUs by 90% was recorded.

The introduction of empty liposomes (table 1, group 2) and a suspension of rhGCSF protein (table 1, group 7) to the irradiated animals led to a slight unreliable increase in the number of CFUs and spleen mass compared with the irradiated control. With the introduction of rhGCSF-containing liposomes, a significant increase in the number of CFUs by 83% was recorded.

Summarizing the above, we can draw the following conclusions

1. According to the endotest indices, the most pronounced positive effects of hematopoietic stem cells in mice irradiated at sublethal doses are observed with the intravenous administration of liposomes containing rhAPP and rhGCSF proteins: an increase in the number of CFUs by 3-3.5 times, spleen masses by 1.5 -3 times in relation to the irradiated control.

2. The combined liposome preparation containing AFP and GCSF can be effectively used for emergency prevention and treatment of acute radiation syndrome.

Compared with the prototype, the claimed method in all its variants provides with different efficacy (optimal in the third embodiment) the possibility of emergency prevention and treatment of acute radiation syndrome.

Claims (3)

1. The method of emergency prevention and treatment of acute radiation syndrome, including the use of a liposomal preparation containing recombinant human alpha protein (rhAFP) in an amount of 0.1-1.0 mg / ml, characterized in that the liposomal preparation is administered intravenously once immediately after exposure in a dose of up to 10 mg / kg body weight in terms of rfAPP or in the same dose according to the scheme: immediately after irradiation and 1, 3, 5, 7 days after irradiation. 2. A method for emergency prevention and treatment of acute radiation syndrome, including the use of a liposomal preparation containing a recombinant human granulocyte colony stimulating factor (rhGKSF) in an amount of 0.004-0.06 mg / ml, characterized in that the liposomal preparation is administered intravenously once immediately after irradiation in a dose up to 0.2 mg / kg body weight in terms of rhGKSF or in the same dose according to the scheme: immediately after irradiation and 1, 3, 5, 7 days after irradiation. 3. A method for emergency prevention and treatment of acute radiation syndrome, including the use of a liposome preparation containing recombinant human alpha protein (rhAFP) in an amount of 0.1-1.0 mg / ml and recombinant human granulocyte colony stimulating factor (rhGKSF) in an amount of 0.004- 0.06 mg / ml, characterized in that the liposomal preparation is administered intravenously once immediately after irradiation at a dose of up to 10 mg / kg body weight in terms of rhAFP and up to 0.2 mg / kg body weight in terms of rhGKSF or the same doses according to the scheme: immediately after about radiation and after 1, 3, 5, 7 days after exposure.