CN115177628A

CN115177628A - Application of cinafenin in preparation of drugs for preventing or treating babesia

Info

Publication number: CN115177628A
Application number: CN202210863012.1A
Authority: CN
Inventors: 李小云; 王锦明; 李余彬; 王彦博; 刘军龙; 赵帅阳; 关贵全; 殷宏; 罗建勋
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2022-07-21
Filing date: 2022-07-21
Publication date: 2022-10-14

Abstract

The invention belongs to the technical field of babesiasis treatment, and particularly relates to an application of cinonide in preparation of a drug for preventing or treating babesiosis. The invention unexpectedly discovers that the cinofenicol can obviously inhibit the growth and the propagation of the sheep babesiosis in vitro, has low toxicity to host cells, can be used as a candidate medicament for preventing and treating the babesiosis and has important significance.

Description

Application of cinafenin in preparation of drugs for preventing or treating babesia

Technical Field

The invention belongs to the technical field of treatment of babesiosis disease, and particularly relates to an application of cinafenin in preparation of a drug for preventing or treating babesiosis.

Background

Babesia disease, also known as tick fever, red urine disease, texas fever, belonging to the piriformis disease. It is a tick-borne hematozoonosis caused by the parasitism of a number of babesia species of the babesiaceae babesia species in erythrocytes of humans or animals.

Generally, animals generally have acute death after being infected by the highly pathogenic babesia, such as some isolates of babesia bovis, babesia ovis and babesia gemmifera; the medium or low pathogenicity babesia species infected with the virus can be of a longer disease period, such as the ovate babesia, the canine babesia, the eastern babesia, the caballi, and the Mohs babesia species. For babesia infecting sheep, the babesia ovis belongs to a highly pathogenic insect species, the babesia morbidii is a pathogen with medium pathogenicity, the body temperature of sick animals is in an audition fever type, the number of red blood cells is reduced, and death occurs in some acute cases; some affected animals may exhibit symptoms of nervous excitement that suddenly die after unintentionally running. The main clinical symptoms after infection are fever, hemolytic anemia, hemoglobinuria, yellow pox, weakness, anorexia and even death.

At present, the babesia infection is generally treated by imidazofenoxuron, benille, trypxanthin, trypan blue, diamidinonazine, imibenea, quinoline urea, artemisinin and the like, but the problems of drug resistance, drug residue of meat products and the like caused by long-term overdose and unreasonable use of drugs exist.

Disclosure of Invention

In view of the above technical problems, an object of the present invention is to provide an application of cinafenin in preparing a drug for preventing or treating babesiosis, which specifically includes the following contents:

in a first aspect, the present invention provides an application of cinafenin in preparing a drug for treating babesia, wherein the structural formula of cinafenin is shown in formula (i) below:

preferably, the babesia species is babesia ovis.

Preferably, the cinafenin is added with pharmaceutically acceptable carriers and/or excipients to prepare any pharmaceutically acceptable dosage form.

Preferably, the dosage form comprises tablets, sprays, granules, capsules, oral liquid, injections and suspensions.

In a second aspect, the present invention provides an application of cinafenin in preparing a drug for preventing babesia, wherein the structural formula of cinafenin is shown in formula (i) below:

preferably, the babesia ovis is a Sinkiang strain of babesia ovis.

Preferably, the cinafentin is added with a pharmaceutically acceptable carrier and/or auxiliary materials to prepare any pharmaceutically acceptable dosage form.

The beneficial effects of the invention are: the invention unexpectedly discovers that the cinofenin (Sinefungin) can obviously inhibit the growth and the propagation of the Babesia ovii in vitro, has low host cell toxicity, can be used as a candidate drug for preventing and treating the Babesia disease, and has important significance for preventing and treating the Babesia disease.

Drawings

FIG. 1 cell proliferation after treatment of MDOK cells with linefungin (Sinefungin);

FIG. 2 shows the cell proliferation after treatment of MDOK cells with Imidocarb dipropinate;

FIG. 3 shows the inhibition of the growth and propagation of Sinkiang strain of Babesia ovis by the cinafenin;

FIG. 4 shows the inhibition of growth and reproduction of Sinkiang sheep Babesia spp.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments. The scope of the invention is not limited to the examples described below.

The experiments described in the following examples obtain biosafety permits and babesiosis laboratory activity permits:

according to the related requirements of biological safety 2-level laboratory (BSL-2) and the biological safety related to babesiosis, the Lanzhou veterinary research institute of China academy of agriculture and sciences reports the biological safety committee, the ethical committee of experimental animals, the biological safety committee of the Chinese academy of sciences, the ethical committee of experimental animals of the Lanzhou veterinary research institute and the biological safety committee of the Lanzhou veterinary research institute step by step, and the Lanzhou veterinary research institute records the requirements of the national biological safety level.

Sources of experimental cells described in the following examples:

sheep kidney epithelial cells MDOK cells and sheep babesia Sinkiang strains were provided by ectoparasites and entomopathogenic team of Lanzhou veterinary institute, national academy of agricultural sciences. Wherein the MDOK cells are cultured in a DMEM (Gibco) complete medium containing 10% FBS (Gibco), the Babesia ovis Sinkiang strain in a RPMI-1640 (Lonza) complete medium containing 20% FBS (Gibco), 5% sheep fresh red blood cells, 37 ℃,5% CO ₂ Culturing in an incubator.

Other reagents in the experiment are common commercial reagents if not specified; the procedures used in the experiments are those known in the art unless otherwise specified.

Example 1 cellular proliferation after treatment of MDOK cells with Cinefungin (Sinefungin)

This experiment was performed in order to evaluate the toxicity of cinafenin (Sinefungin) to host cells. Adding 100 μ L DMEM complete medium containing MDOK cells into a 96-well plate, incubating for 2-3 h until the cells adhere to the wall, discarding the medium, adding 100 μ L complete medium containing different concentrations of cinafenin (Sinefungin) (0.125 μ M, 0.625 μ M, 3.125 μ M, 6.25 μ M, 12.5 μ M, 25 μ M, 50 μ M, 100 μ M), setting 3 replicates for each concentration gradient, positive control imidazopheniramide (Imidocupropinate) (0.125 μ M, 0.625 μ M, 3.125 μ M, 6.25 μ M, 12.5 μ M, 25 μ M, 50 μ M, 100 μ M), blank control adding complete medium without drug treatment, culturing for 3 days, changing medium for 24h and supplementing with drugs at corresponding concentrations, respectively, adding 100 μ L complete medium containing CCK-728 μ K, and using ELISA measuring instrument when the medium is at 24h,48h, 7210 μ M, and adding the complete medium containing CCK-3 μ M. And drawing an inhibition rate fitting curve by using Graphpad prism 8, and finding out the corresponding drug concentration when the inhibition rate is 50%.

Inhibition = (blank control-drug treated group)/(blank control-background value) × 100%.

The cell proliferation results of the MDOK cells treated by the cinafenin (Sinefungin) are shown in fig. 1, and the toxicity of the cinafenin (Sinefungin) to the cells is stronger with the increase of the concentration of the cinafenin (Sinefungin), but the inhibition rate of the cinafenin (Sinefungin) to the cells is less than 25% at each time point when the concentration reaches 100 μ M, namely, the cinafenin (Sinefungin) is less toxic to the host cells.

The results of the cell proliferation after treatment of MDOK cells with Imidocarb diprinate are shown in fig. 2, and the increase of the Imidocarb diprinate concentration results in stronger toxicity to cells, and the inhibition rate of the Imidocarb diprinate concentration at 25 μ M is over 50%, which is obviously more toxic to cells than that of cinafenin (Sinefungin).

Example 2 inhibition of Sinofenin (Sinefungin) growth and reproduction of Sinkiang sheep Babesia in vitro

This test was performed to evaluate the inhibition of different concentrations on the growth and reproduction of Sinkiang sheep Babesia. Various concentrations of cinafenin (0.9375 μ M, 1.875 μ M, 3.75 μ M, 7.5 μ M, 15 μ M, 30 μ M) diluted in medium were added to 24-well plates containing 2ml of complete medium at a final bayberry worm staining rate of 0.2%, each concentration was 3 replicates, the positive controls were imidazophenylurea (12.5 nM, 25nM, 50nM, 100nM, 200nM, 400 nM), since the solvent of cinafenin (Sinefungin) was water, no negative control was provided, the blank control was not treated with drug, and placed in an incubator for 4 days. Blood smears were sampled at 48h,72h and 96h, fixed in methanol, stained with Gimesas, photographed microscopically and the rate of staining calculated. And drawing an inhibition rate fitting curve by using Graphpad prism 8, and finding out the corresponding drug concentration when the inhibition rate is 50%.

Inhibition = (blank control-drug treated group)/blank control group × 100%.

The results of the in vitro inhibition of the growth and propagation of Sinkiang Babesia ovis strain by Simefungin (Sinefungin) are shown in figure 3, the Sinkiang Babesia ovis strain is treated by the Simefungin (Sinefungin) with different concentrations, when the concentration is increased, the growth inhibition effect on Babesia ovis is more obvious, and the half Inhibition Concentration (IC) is ₅₀ ) About 2.5. Mu.M.

The results of the imidazole phenylurea (Imidocabron dipropinate) for inhibiting the growth and the reproduction of the Sinkiang Babesia ovis strain in vitro are shown in figure 4, the imidazole phenylurea (Imidocabron dipropinate) with different concentrations is used for treating the Sinkiang Babesia ovis strain, when the concentration is increased, the growth inhibition effect on Babesia is more obvious, and the half Inhibition Concentration (IC) is more obvious ₅₀ ) Between 25nM and 50 nM.

In conclusion, in the experimental process, the invention unexpectedly discovers that compared with imimidazophenylurea, the cinafenin has a better inhibition effect on babesia, is low in cytotoxicity and better in safety, and can be used for preventing or treating the babesiosis.

The above embodiments are merely preferred embodiments of the present invention, and not intended to limit the scope of the invention, so that equivalent changes or modifications made based on the structure, characteristics and principles of the invention should be included in the claims of the present invention.

Claims

1. The application of the cinafenin in preparing the drugs for treating babesia is characterized in that the structural formula of the cinafenin is shown as the following formula (I):

2. the use of claim 1, wherein the babesia is babesia ovis.

3. The use according to claim 1 or 2, wherein the cinafenin is formulated with pharmaceutically acceptable carriers and/or excipients into any pharmaceutically acceptable dosage form.

4. The use of claim 3, wherein the dosage form comprises a tablet, spray, granule, capsule, oral liquid, injection, suspension.

5. The application of the cinafenin in preparing the drugs for preventing babesia is characterized in that the structural formula of the cinafenin is shown as the following formula (I):

6. the use of claim 5, wherein the Babesia spp.

7. The use according to claim 5 or 6, wherein the cinafenin is formulated with pharmaceutically acceptable carriers and/or excipients into any pharmaceutically acceptable dosage form.

8. The use of claim 7, wherein the dosage form comprises tablets, sprays, granules, capsules, oral liquids, injections, suspensions.