RU2843185C1 - Method for assessing antileukemic activity of preparations - Google Patents

Abstract

FIELD: veterinary immunology.

SUBSTANCE: invention refers to veterinary immunology, namely to a method for assessing antileukemic activity of preparations. Method for evaluating antileukemic activity of preparations, including the isolation of a cell culture, the introduction of a virus-containing suspension into a supporting medium, its titration in 96-well immunological plates by the method of double serial dilutions, introduction into the experimental rows of holes of the test preparations in different concentrations, incubation of plates in a thermostat, determination of virus titre and virus inhibition coefficient, characterized by the fact that a lymphocyte culture from freshly obtained peripheral blood from a cow with leukaemia is introduced into the supporting medium, incubation is carried out for 24 hours, by means of immunofluorescence reaction on 4-cross system establishing minimum inhibitory concentration of drug by virus titre, and in order to determine the inhibition coefficient (Ki), the obtained titre values are converted to logarithms with base 2, high antileukemic activity of the preparation is noted if the values Ki are from 100 to 63.5, the average one is from 36.3 to 63.49, the low one is at values up to 36.29.

EFFECT: invention enables rapid simultaneous testing of a large number of chemical compounds and has high reliability.

1 cl, 4 tbl, 3 ex

Description

The invention relates to veterinary immunology, namely to laboratory research methods, and can find application in predicting the prophylactic and/or immunotherapeutic effectiveness of various chemical compounds in leukemic infection in vitro.

Bovine leukemia virus (BLV) is an oncogenic retrovirus belonging to the genus Deltaretrovirus (family Retroviridae) that predominantly infects B lymphocytes and causes persistent infection with variable clinical outcomes in cattle. The disease is widespread throughout the world, causing significant economic losses associated with reduced milk production, the inability to export animals, semen and embryos [Bartlett P.C., Ruggiero V.J., Hutchinson H.C., Droscha C.J. et al. Current developments in the epidemiology and control of enzootic bovine leukosis as caused by bovine leukemia virus // Pathogens. - 2020. - Vol. 9(12). - P. 1058.]. In recent years, there has been increasing evidence of the presence of the virus in humans, supporting the hypothesis that BLV is a zoonotic agent [Olaya-Galán N.N., Blume S., Tong K., Shen H., Gutierrez M.F., Buehring G.C. In vitro susceptibility of human cell lines infection by bovine leukemia virus // Front. Microbiol. - 2022. - Vol. 13. - A. 793348].

Due to the lack of effective treatment and vaccination methods, the fight against leukemia infection is based on preventive measures aimed at reducing the transmission of the virus, which do not always give a positive result, since they require huge economic costs for their implementation [Rodríguez S.M., Florins A., Gillet N., de Brogniez A. et al. Preventive and therapeutic strategies for bovine leukemia virus: lessons for HTLV // Viruses. 2011. 3(7). P. 1210-48.]. Therefore, a relevant area of research is the search for new chemical compounds that effectively reduce the level of antiviral load in BLV infection, as well as methods for monitoring their effectiveness.

A method for determining the sensitivity of tumor lymphocytes to chemotherapeutic drugs is known [Kravchenko D.V., Svirnovsky A.I. Chronic lymphocytic leukemia: clinical features, diagnostics, treatment. - Gomel: State Institution "Republican Scientific and Practical Center for Cancer and Hepatitis C", 2017. - P. 55-62]. The method is intended for monitoring the formation of drug resistance of cells to individual drugs and their combinations during treatment, which makes it possible to predict the response to subsequent therapy and, in cases of drug insensitivity, promptly change them for a specific patient based on a study of the drug sensitivity of his cells in a test with methylthiazole tetrazolium bromide (MTT). The disadvantage of this method is the duration of the research (48 hours of cell cultivation in a nutrient medium with the necessary additives, then 44-hour incubation with the tested drugs and an additional 4-hour exposure with MTT), as well as the labor intensity and high cost of the reagents required to set up the reaction.

Preclinical studies of new chemical compounds, including the assessment of their antileukemic activity, can also be carried out by experimentally infecting guinea pigs with VLKRS [Patent RU 2802150 C1 Method for modeling leukemic infection in animals. Patent holder: FGBNU "Omsk Scientific Research Center", published on 22.08.2023, Bulletin No. 24]. The method is suitable for studying the specific antiviral activity of various chemical compounds, biologically active substances and immunomodulators, while monitoring the effectiveness of the prophylactic or therapeutic effect of the test drugs in vivo can be carried out using the immunofluorescence reaction (IF) [Mironov A.N., Bunyatyan N.D. et al. Guide to conducting preclinical studies of drugs. Part one. - M.: Grif i K, 2012. - P. 533-535]. The evaluation of the antiviral action of substances, especially when simultaneous testing of several derivatives of a chemical compound is necessary, in experimental viral infection on animals has disadvantages associated with the need to use a large number of guinea pigs in the experiment, as well as with the duration of obtaining experimental data.

The closest technical solution to the claimed one is the method of studying antiviral drugs in cell culture using the micromethod, which provides for the following: growing cell cultures in 96-well plastic plates; adding 0.1 ml of virus-containing suspension to each well; 0.1 ml of supporting medium containing the substances under study with a dilution factor of 1:2, starting with a concentration of 1000 μg/ml; microscopy of cell cultures to determine the titer by cytopathic effect and calculate the selectivity index of the drugs [Mironov A.N., Bunyatyan N.D. et al. Guide to Conducting Preclinical Studies of Drugs. Part One. - Moscow: Grif i K, 2012. - P. 526]. Despite the fact that this method allows for the simultaneous study of a large number of compounds with minimal consumption of nutrient media and substances under study, it, like the previous method, is lengthy to set up and also requires many hours of observing the cells under a microscope.

The technical result of the invention is the creation of a method for assessing the antileukemic activity of drugs based on the immunofluorescence method, which allows for the rapid simultaneous testing of a large number of chemical compounds with minimal material costs and has high reliability.

The technical solution for the method of assessing the antileukemic activity of drugs is to isolate a lymphocyte culture from freshly obtained peripheral blood from a cow sick with leukemia using centrifugation in a density gradient of a radiopaque substance (trasograph or its analogue), adding 100 μl of the supporting medium (Hanks' solution) to the wells of a 96-well plate for immunological studies, adding 100 μl of the virus-containing suspension to the first well of each row, obtaining a 1:2 dilution, then 100 ml from the first wells from the 1:2 dilution are added to the second wells of each row, obtaining a 1:4 dilution, a 1:8 dilution is prepared from this dilution, transferring 100 μl of liquid from one well, then dilutions of 1:16, 1:32, etc. are prepared by the method of successive dilutions. Excess liquid of 100 μl is removed from the wells of the last row (1:4096). Then 100 μl of Hanks' solution is added to the wells of the 1st (control) row, and 100 μl of different concentrations of the test drug diluted in Hanks' solution are added to the wells starting from the 2nd row and ending with the 5th row (test rows). For this purpose, 1000 mg of the drug is taken and diluted in 1 ml of Hanks' solution and a series of successive 10-fold dilutions are made: 100, 10 and 1 mg/ml. In the cell suspensions located in the wells of the control row, the virus activity is assessed by titer before incubation and after 24-hour incubation in a thermostat at a temperature of 37°C in plates with a closed lid placed in a desiccator to create conditions of reduced air access by preparing smears of the "dried drop" type and setting up an immunofluorescence reaction (RIF) [Patent RU 2810589 C1 Method for setting up an immunofluorescence reaction for the diagnosis of bovine leukemia. Patent holder FGBNU "Omsk Scientific Research Center", published on 27.12.2023, Bulletin No. 36]. In the cell suspensions of the experimental rows (from the 2nd to the 5th rows), the virus activity by titer is carried out only after 24-hour incubation. To assess the intensity of luminescence, a four-cross system is used: (++++) - very bright luminescence, clearly contrasting against a dark background; (+++) - bright luminescence; (++) or (+) - weak luminescence; (-) - no luminescence. A positive reaction is considered to be luminescence in three or four crosses. The minimum inhibitory concentration of the drug is established based on the virus titer, then the obtained titer values are converted to logarithms with a base of 2 (a virus titer of 1:2 is taken to be equal to 1.0, 1:4 - 2.0, etc.) and the inhibition coefficient ( _Ki ) of the virus is determined using the formula

K _i = ((A _k - A ₀ )/A _k ) * 100,

where A _k is the virus titer in the control series (without adding the studied agent to the support medium);

A _o is the titer of the virus in the experimental series (with the addition of the test drug to the support medium).

Depending on the concentration of the drug, high anti-leukemic activity is observed at _K values from 100 to 63.5, average - from 36.3 to 63.49, low - at values up to 36.29.

The distinctive features of the proposed method are: the use of a culture of peripheral blood lymphocytes with a high concentration of VLCRS, 24-hour incubation of the virus-containing suspension in a support medium and an assessment of the activity by the titer of the virus in the RIF with subsequent calculation of the virus inhibition coefficient.

The essence of the invention is explained using specific examples of the method implementation.

Example 1. A test of the antileukemic activity of several drugs with potential antiviral activity is carried out using the proposed method:

- betulin (pentacyclic triterpenoid of the lupane group, has a wide range of biological activity, including antiviral);

- dextranal (a natural biopolymer obtained by modifying dextran, antiviral activity has not been studied);

- fullerene C60 (allotropic molecular form of carbon).

The evaluation of the virus activity by titer using RIF in the control (without the drug) and experimental rows (with the tested drugs) of wells is carried out using different incubation exposures: 3, 6, 24 and 48 hours, and in the control row of wells also before the start of incubation.

The results of the research are presented in Table 1.

Table 1 - Minimum inhibitory titers of the virus in RIF at different times after incubation of peripheral blood lymphocyte culture with different drugs

Preparation Concentration of the drug in Hanks' solution, mg/ml Incubation time, hours 3 6 24 48 Betulin 1000 1:256 1:64 1:32 - 100 1:512 1:512 1:512 - 10 1:1024 1:1024 1:1024 - 1 1:2048 1:1024 1:1024 - Dextranal 1000 1:64 1:32 - - 100 1:128 1:64 1:32 1:32 10 1:512 1:512 1:512 1:64 1 1:2048 1:1024 1:1024 1:128 Fullerene 1000 1:64 1:32 1:32 - 100 1:128 1:128 1:128 - 10 1:256 1:128 1:16 1:16 1 1:512 1:128 1:128 1:32 Post-incubation control Hanks solution without drug 1:2048 1:2048 1:2048 1:256 Pre-incubation control 1:2048

As can be seen from Table 1, in the control rows of wells both before and after incubation for 3, 6, and 24 hours, the virus activity in RIF was the same (virus titer 1:2048). After 48-hour incubation, the virus titer dropped to 1:256, indicating a significant decrease in its activity and, therefore, the inappropriateness of testing chemical compounds under such exposure.

The most pronounced anti-leukemic activity of the tested preparations betulin and dextranal was demonstrated when they were incubated at the maximum concentration (1000 mg/ml) with a virus-containing suspension for 24 hours, when using which by this time the virus titer reached a minimum or complete suppression of viral activity occurred. At lower concentrations, the antiviral effectiveness of the preparations decreased.

Fullerene, unlike other compounds, showed antileukemic activity at a concentration of 1000 mg/ml and especially at a concentration of 10 mg/ml during 24-hour incubation. At a concentration of 1.0 mg/ml, this preparation also had an antiviral effect, as indicated by a virus titer of 1:128.

Thus, the most appropriate method is 24-hour incubation of the test preparations in a supportive medium, since such exposure allows for the most reliable assessment of their anti-leukemic activity.

It should also be noted that multiple studies of isolated peripheral blood lymphocytes from different cows with leukemia in RIF (before incubation) showed approximately the same result, with the average virus titer being 1:2048, and the highest value being 1:4096.

In the future, for a quantitative assessment of the degree of anti-leukemic activity, the values of virus titers established by us during 24-hour exposure of the drugs are converted into logarithms with a base of 2 (the virus titer 1:2 is taken to be equal to 1.0, 1:4 - 2.0 ... 1:32 - 5.0, etc.) and the virus inhibition coefficient is calculated using the formula

K _i = ((A _k - A ₀ )/A _k ) * 100,

where A _k is the virus titer in the control series (without adding the studied agent to the support medium);

A _o is the titer of the virus in the experimental series (with the addition of the test drug to the support medium).

For example, let us substitute into the formula the values obtained using betulin at a concentration of 1000 mg/ml. The established virus titer of 1:32 is taken to be 5 when converted to logarithm, and in the control - 1:2048 is taken to be 11.

K _i = ((11- 5)/11) * 100 = 54.5

The inhibition coefficient was 54.5.

When using betulin at a concentration of 100 mg/ml _{, K} is 18.2, and at concentrations of 10 and 1.0 mg/ml - 9.1 (Table 2).

Table 2 - Virus inhibition coefficients when using drugs in different concentrations

Preparation Concentration of the drug in Hanks' solution, mg/ml Inhibition coefficient Betulin 1000 54.5 100 18.2 10 9.1 1 9.1 Dextranal 1000 100,0 100 100,0 10 18.2 1 9.1 Fullerene 1000 54.5 100 36.4 10 63.6 1 36.4

As shown in Table 2, dextranal had the highest antileukemic efficacy, with K _i equal to 100.0 at concentrations of 1000 and 100 mg/ml, followed by fullerene at a concentration of 10 mg/ml (K _i = 63.6). The average level of suppressive efficacy against BLV was demonstrated by betulin at a concentration of 1000 mg/ml and fullerene at concentrations of 1000, 100, and 1.0 mg/ml (K _i from 36.4 to 54.5).

Example 2. The antileukemic activity of new chemical compounds is tested in vitro and in vivo. For this purpose, two structural modifications of 3-acetylpyridine derivatives (compounds I and II) are taken, some compounds of which have pronounced antiviral activity [Stalinskaya A.L., Martynenko N.V., Shulgau Z.T., Shustov A.V., Keyer V.V., Kulakov I.V. Synthesis and antiviral properties against SARS-CoV-2 of epoxybenzooxocino[4,3-b]pyridine derivatives // Molecules. - 2022. - Vol. 27(12). - P. 3701].

In vitro antiviral activity study.

When testing antileukemic activity using the proposed method (in vitro), rimantadine hydrochloride (a drug used to treat and prevent influenza) is used as a known comparison drug.

The results of the research are presented in Table 3.

Table 3 - Minimum inhibitory titers of the virus in RIF and inhibition coefficients after 24-hour incubation of peripheral blood lymphocyte culture with the tested drugs

Preparation Concentration of the drug in Hanks' solution, mg/ml Virus titer in RIF Inhibition coefficient Compound I 1000 - 100,0 100 1:8 72.7 10 1:256 27.3 1 1:256 27.3 Compound II 1000 1:16 63.6 100 1:256 27.3 10 1:256 27.3 1 1:256 27.3 Rimantadine hydrochloride 1000 1:32 54.5 100 1:256 27.3 10 1:512 9.1 1 1:2048 0 Post-incubation control Hanks solution without drug 1:2048 - Pre-incubation control 1:2048 -

As shown in Table 3, compound I had the highest antileukemic activity at concentrations of 1000 and 100 mg/ml (K _and 100.0 and 72.7, respectively), as well as compound II at the maximum concentration (K _and 63.6). When incubated with a virus-containing suspension of rimantadine hydrochloride, a weaker inhibitory efficiency was observed.

Study of antiviral activity in vivo.

25 guinea pigs are selected and divided into 5 groups of 5 individuals each. Animals of the first 4 groups are infected intraperitoneally with a cell suspension of lymphocytes isolated from a cow sick with leukemia in the hematological stage, in a volume of 1 ml [Patent RU 2802150 C1 Method for modeling leukemia infection in animals. Patent holder FGBNU "Omsk Scientific Research Center", published 08/22/2023, Bulletin No. 24]. Individuals of the 1st group are administered compound (I) once 2 hours before infection, intraperitoneally, at a dose of 30 mg / kg (1: 8 LD ₅₀ ); The 2nd group is administered compound (II) intraperitoneally once 2 hours before infection at a dose of 15 mg/kg (1:8 LD ₅₀ ), the 3rd group is administered rimantadine hydrochloride in the same way at a dose of 15 mg/kg (1:8 LD ₅₀ ). The guinea pigs of the 4th group are not administered the drug (positive control). The remaining 5 individuals (the 5th group) are intact and serve as a negative control. The infectious status of the animals is assessed by blood testing using RIF on the 30th and 90th days after infection.

The results of the research are presented in Table 4.

Table 4 - Anti-leukemic activity of the test compounds in guinea pigs in RIF

Group of animals Study period after infection, days 30 90 1st group 1/5 1/5 2nd group 1/5 0/5 3rd group 5/5 5/5 4th group 5/5 5/5 Control 0/5 0/5

Note: numerator is the number of animals that responded positively; denominator is the number of animals in the group.

As shown in Table 4, compounds I and II had the highest antileukemic activity, as indicated by the absence of specific luminescence in the RIF on the 30th and 90th days after infection of guinea pigs with BLV in 80-100% of animals. In those treated with rimantadine, as well as in individuals of the 4th group, which were not administered the studied preparations after virus inoculation, immunofluorescence of cells was noted in 100% of cases.

Thus, high anti-leukemic efficacy of compounds I and II was established both in vitro and in vivo, which indicates the high reliability of the proposed method.

It should be noted that when using peripheral blood lymphocytes isolated from a cow with leukemia, it becomes possible to obtain a high concentration of the virus (the titer of the virus in the RIF is 1:2048), while there is no need for long-term incubation of the virus-containing suspension in the cell culture to wait for the maximum accumulation of the virus. In addition, the proposed method is cost-effective, since it eliminates the use of some expensive reagents, and also allows for a preliminary simultaneous assessment of the antileukemic activity of a number of chemical compounds and their derivatives to select the most promising drugs for further studies on laboratory animals. A high degree of coincidence of the results of the study of the antileukemic activity of drugs in vitro and in vivo indicates the reliability of the proposed method.

Claims (1)

Method for assessing the anti-leukemic activity of drugs, including the isolation of cell culture,introducing a virus-containing suspension into the support medium, titrating it in 96-well immunological plates using the method of two-fold serial dilutions, introducing test preparations in different concentrations into the experimental rows of wells, incubating the plates in a thermostat, determining the titer of the virus and the coefficient of inhibition of the virus, characterized in that a lymphocyte culture from freshly obtained peripheral blood from a cow sick with leukemia is introduced into the support medium, incubation is carried out for 24 hours, using the immunofluorescence reaction according to the 4-cross system, the minimum inhibitory concentration of the preparation is established based on the titer of the virus, and to determine the coefficient of inhibition (K_And) received titer values are converted to logarithms with a base of 2, high anti-leukemic activity of the drug is noted at K values_Andfrom 100 to 63.5, medium - from 36.3 to 63.49, low - with values up to 36.29.