Application of cordycepin for treating babesia infection and composition of cordycepin

Abstract

The invention relates to the field of medicines, and discloses application of cordycepin to preparation of a medicine for treating babesia infection and a pharmaceutical composition of the cordycepin. The invention relates to application of cordycepin in preparation of a medicine for treating babesia infection. The composition contains a therapeutically effective amount of cordycepin and pharmaceutically acceptable carriers or auxiliary materials, can be prepared into dosage forms such as tablets, granules, capsules, oral liquid, injections or suspensions, and is suitable for administration modes such as oral administration. In-vitro evaluation shows that the cordycepin has inhibitory activity on Babesia bovis and Babesia gibsoni, and the median inhibitory concentrations (Ic50) of the cordycepin and the Babesia gibsoni are respectively about 303.8 Nm and 147.5 Nm; in a mouse babesia vole infection model, the parasitic rate peak value can be obviously reduced and delayed through oral administration of 20 Mg / Kg (the model group is about 17.5% / 13th day, and the cordycepin group is about 1.8% / 17th day). A cytotoxicity test (Hff-1, Cck-8) shows that the inhibition rate on cell proliferation under the condition of 100 microns is less than 10%. The cordycepin and the pharmaceutical composition thereof can be used for preventing and / or treating babesia infection.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and more particularly to the use of cordycepin in the preparation of medicaments for treating Babesia infection and pharmaceutical compositions thereof. Background Technology

[0002] Babesia are protozoa that parasitize the red blood cells of vertebrates, causing babesiosis in animals and posing a risk of cross-species transmission. Babesia gibrini, Babesia bovis, and Babesia vole are among the more common species found in clinical and veterinary settings. Infection primarily manifests as fever, hemolytic anemia, jaundice, and hemoglobinuria. Severe cases can lead to organ damage, placing a burden on livestock farming and public health.

[0003] Current treatment primarily involves combination chemotherapy. Commonly used clinical and laboratory regimens include combinations of atovaquinone and macrolides (such as azithromycin), as well as combinations of lincosamides and quinine; veterinary medications also include diamine pyrimidines such as diaminoethylamidine acetate. The efficacy of these drugs varies among different hosts and different Babesia species, and long-term or repeated use carries a risk of relapse and drug resistance. Furthermore, some drugs and regimens are associated with safety issues such as hepatotoxicity, nephrotoxicity, bone marrow suppression, and gastrointestinal adverse reactions, limiting their application in chronically or immunocompromised hosts. For human use, the limited variety of available drugs and constraints on price and accessibility are also practical problems.

[0004] From the perspective of mechanism of action, anti-Babesia drugs mainly focus on pathways such as mitochondrial electron transport, folic acid, or nucleic acid metabolism. Nucleoside (acid) analogs have a theoretical advantage due to their ability to interfere with protozoan nucleic acid synthesis, but there is a shortage of available lead compounds for Babesia, and some molecules also face bottlenecks such as poor in vivo stability, unclear selectivity, or dose-related adverse reactions. On the other hand, pharmaceutical research around the active ingredients is relatively weak, lacking compositions and formulation pathways that take into account dissolution, absorption, stability, and production feasibility, making it difficult to meet the demand for drugs that are "highly effective, low in toxicity, easy to manufacture, and scalable."

[0005] In summary, there is an urgent need for existing technologies to provide an active ingredient and its pharmaceutical composition with a well-defined structure, good inhibitory potential against Babesia, and superior safety, and to establish formulations suitable for oral or injectable administration to improve therapeutic efficacy, reduce the risk of relapse and drug resistance, and improve accessibility in clinical and veterinary fields. Summary of the Invention

[0006] The purpose of this invention is to provide an effective active ingredient and its pharmaceutical composition for treating Babesia infection, specifically cordycepin (also known as 3′-deoxyadenosine, molecular formula C). 10 H 13The use of N5O3 in the preparation of drugs for treating Babesia infection, and pharmaceutical compositions containing the active ingredient to address the problems of unstable efficacy, poor tolerability and accessibility of existing drugs.

[0007] To achieve the above objectives, the present invention proposes the following technical solution: Cordycepin is used to prepare a drug for treating Babesia infection; the drug is suitable for infections caused by Babesia gibrini, Babesia bovis, or Babesia vole. The drug can be formulated into any dosage form, such as tablets, granules, capsules, oral liquids, injections, or suspensions, and can be administered via pharmaceutically acceptable routes such as oral administration. Furthermore, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of cordycepin and a pharmaceutically acceptable carrier or excipient for treating Babesia infection.

[0008] The technical effects of this invention are as follows: Cordycepin exhibits clear inhibitory activity against Babesia, with effective inhibition observed in vitro at approximately 1 μmol / L; in a mouse babesia infection model, administration at 20 mg / kg body weight significantly reduces the infection rate, demonstrating good in vivo inhibitory effects; cytotoxicity assessment shows minimal impact on host cell proliferation at higher concentrations (up to 100 μm), suggesting good tolerability. Compared with existing technologies, the active ingredient and pharmaceutical composition provided by this invention have well-defined structures, definite effects, and a clear formulation pathway, making them suitable for further development into drugs for treating Babesia infection. Attached Figure Description

[0009] Figure 1 : These are the preliminary screening results of the candidate compounds' in vitro inhibition of Babesia gibrini.

[0010] Figure 2 : Results of Hff-1 cell proliferation / viability after treatment with different concentrations of cordycepin.

[0011] Figure 3 The figures show the in vitro inhibition curves and half-maximal inhibitory concentration (IC50) fitting results of cordycepin against Bovine Babesia and Babesia gibrini.

[0012] Figure 4 : Parasitism rate-time curve of mouse babesiosis infection model.

[0013] Figure 5 : Morphological observation of blood smears from mice in each treatment group (100× oil immersion).

[0014] Note: In continuous in vivo observations, a trend of reduced and delayed host cell damage associated with Toxoplasma gondii infection was observed; this phenomenon is an illustrative result of the second technical point of this application. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] The present invention will be further described in detail below with reference to specific embodiments.

[0017] The preliminary in vitro screening results of *Babesia gibrini* by the Central Tibetan Medicine Library showed that 21 effective drugs were identified at a drug concentration of 10 μm. Eleven effective drugs were identified at a drug concentration of 1 μm. Figure 1 As shown.

[0018] Example 1: Cell proliferation of Hff-1 cells after cordycepin treatment

[0019] This experiment was conducted to evaluate the toxicity of cordycepin to host cells. 100 μl of Dmem complete medium containing Hff-1 cells was added to a 96-well plate, with a cell count of 1 × 10⁻⁶ cells. 4 Cells were incubated per well for 24 h. 100 μL of different concentrations of cordycepin dilution (100 μm, 50 μm, 10 μm, 1 μm, 500 nm, 100 nm, 10 nm) were added to each well, with four replicates for each gradient. A positive control was provided using benzir (Da) (100 μm, 50 μm, 10 μm, 1 μm, 500 nm, 100 nm, 10 nm), and a blank control was provided using 100 μL of complete medium containing 0.5% DmSO. Cells were incubated for 24 h. After 24 h, 10 μL of Cck-8 solution (Cck-8 kit) was added to each well, and the cells were incubated in the same incubator for 0.5 h. The absorbance was measured at 450 nm, and cell viability was calculated according to the manufacturer's instructions.

[0020] The results of Hff-1 cell proliferation after cordycepin treatment are as follows: Figure 2 As shown, the toxicity of cordycepin to cells increases with increasing concentration, but when the concentration reaches 100 μm, the inhibition rate of cordycepin on cells is less than 10% at each time point, indicating that cordycepin has low toxicity to host cells.

[0021] Example 2: In vitro inhibition of bovine babesia and gibbese babesia growth and reproduction by cordycepin

[0022] The test compound was diluted in complete culture medium to obtain eight desired concentrations (2-fold serial dilutions). Next, the synchronic parasites were treated with 5% D-fructose to obtain ≥90% circular parasites. Then, 50 μl of synchronic parasites were inoculated into 96-well plates containing 50 μl of the test compound to achieve a parasite infection rate of 0.5% and a hematocrit of 2%.

[0023] To evaluate the inhibitory effects of different concentrations of cordycepin on the growth and reproduction of *Babesia bovis* and *Babesia gibrevicornu*, such as... Figure 3 As shown, this experiment was conducted. Different concentrations of cordycepin (100 μm, 50 μm, 10 μm, 1 μm, 500 nm, 100 nm, 10 nm) diluted with culture medium were added to 96-well plates containing 2 ml of complete culture medium with a final Babesia bovis or Babesia gibrevicornu infection rate of 0.5% and a hematocrit of 2%. Each concentration was replicated in triplicate. The positive control was benziridine (100 μm, 50 μm, 10 μm, 1 μm, 500 nm, 100 nm, 10 nm), the negative control was 1% dimethyl sulfoxide, and the blank control was untreated. The 96-well plates were incubated at 37°C and 5% Co2 for 3 days. Then, 100 μl of lysis buffer containing 0.02% (v / v) Sybr Green I was added to each well, mixed well, and the 96-well plates were incubated at room temperature in the dark for 1 hour. Then, fluorescence intensity was measured using a visible light nucleic acid detection instrument at excitation and emission wavelengths of 485 nm and 518 nm, respectively. An inhibition rate fitting curve was plotted using Graphpadprism8 to find the drug concentration corresponding to an inhibition rate of 50%.

[0024] Inhibition rate = (blank control group - drug treatment group) / blank control group × 100%.

[0025] Results of cordycepin's in vitro inhibition of Babesia growth and reproduction: Figure 2 As shown, different concentrations of cordycepin were used to treat *Babesia bovis* or *Babesia gibrini*. The higher the concentration, the more significant the inhibitory effect on *Babesia* growth, with the half-maximal inhibitory concentration (IC50) reaching [value missing]. 50 The wavelengths were 303.8 nm (Babesia bovis) and 147.5 Nm (Babesia gibrini), respectively.

[0026] Example 3: In vivo efficacy of cordycepin against Babesia babesiosis in voles

[0027] Twenty healthy female Balb / c mice were randomly divided into three groups: a control group treated with 15 mg / kg atovaquinone + 20 mg / kg azithromycin, a Babesia infection model group, and a 20 mg / kg cordycepin treatment group. All mice in the model and treatment groups were intraperitoneally injected with 1×10 mg of cordycepin. 7One *Babesia* parasite was recorded as day 0. When the average erythrocyte parasite infection rate in each group of mice reached 1% (day 4), mice in the atovaquinone + azithromycin treatment group and the 20 mg / kg cordycepin treatment group were administered the drug by gavage once daily at the same time for 5 consecutive days. Starting from day 1, blood was collected from the tail vein of all mice every 2 days. Giemsa-stained blood smears were used to examine for parasitemia, and the infection rate was calculated. Observations were continuously recorded until day 31, and a erythrocyte parasite infection rate curve was plotted. Figure 3 As shown, the infection rate of mice in the Babesia babesiosis model group peaked at 17.5% on day 13 post-infection; while the infection rate in the cordycepin-treated group peaked at only 1.8% on day 17. On day 13 post-infection, compared with the control group, cordycepin significantly (P<0.05) inhibited the infection rate in mice. These results indicate that cordycepin has a good therapeutic effect on acute Babesia babesiosis in voles. It also suggests that cordycepin may alleviate and delay the damage caused by Toxoplasma gondii to the host in mice, such as… Figure 4 As shown.

[0028] To evaluate the effects of cordycepin on Babesia proliferation and its protective effect on mouse erythrocytes, the parasites and erythrocytes in blood smears were observed using a 100x oil immersion microscope. The results are as follows: Figure 4 , Figure 5 As shown, starting 7 days after infection with Babesia villiformis, the number of Babesia cypriniformes produced by fission in the erythrocytes of mice in the control group was significantly increased compared to the cordycepin-treated group. On day 13 of infection, the erythrocytes of mice in the control group showed obvious swelling, deformation, uneven staining, and unequal size. The erythrocytes of mice in the cordycepin-treated group had essentially normal morphology, with no severe pathological damage, and were largely consistent with the erythrocytes of the blank control group. These results indicate that cordycepin can inhibit the proliferation of Babesia villiformis in host cells and alleviate the damage to erythrocytes caused by Babesia villiformis infection.

[0029] Compared with commonly used drugs such as amikacin, clindamycin, and atovaquinone, cordycepin (Cor) showed stronger inhibitory activity against Babesia growth in in vitro experiments, with an inhibition rate of over 85% (Ic). 50 The value is significantly lower than that of traditional drugs, and it works rapidly, with a noticeable decrease in parasitic malnutrition within 24 hours.

[0030] Existing anti-Babesia drugs generally have adverse reactions such as hepatotoxicity, nephrotoxicity, bone marrow suppression, or allergic reactions, limiting their long-term or high-dose use. This invention, through cytotoxicity experiments (such as Cck-8 and Ldh release assays) and acute animal toxicity tests, verifies that cordycepin, within its effective anti-parasitic concentration range, has no significant toxicity to mammalian cells (such as Hff) or experimental animals, and Ld... 50It is significantly higher than the therapeutic dose, has a high therapeutic index (Ti), and a large safety window. It is particularly suitable for long-term intervention in people with weakened immune systems or chronic infections.

[0031] Furthermore, cordycepin can be extracted from Cordyceps militaris or obtained through microbial fermentation or chemical synthesis. Compared to natural drugs that rely on rare plant or animal sources, its raw material sources are widespread and highly sustainable. At the same time, its structure is easily chemically modified, providing a good foundation for the subsequent development of derivatives with more active or superior pharmacokinetic properties. It possesses good druggability and scalability, which is conducive to achieving low-cost, large-scale production and promoting industrialization.

[0032] The above content is merely an example and illustration of the present invention. Any modifications, substitutions, combinations, simplifications, etc., made by those skilled in the art to the specific implementation described herein are equivalent substitutions and are all included within the protection scope of the present invention.

Claims

1. Use of cordycepin in the preparation of drugs for the treatment of Babesia infection.

2. The use of cordycepin according to claim 1 in the preparation of a medicament for treating Babesia infection, characterized in that: The cordycepin is 3′-deoxyadenosine.

3. The use of cordycepin according to claim 1 in the preparation of a medicament for treating Babesia infection, characterized in that: The molecular formula of cordycepin is as follows: .

4. The use of cordycepin according to claim 1 in the preparation of a medicament for treating Babesia infection, characterized in that: The babesi is selected from one or more of Babesia gibrini, Babesia bovis, and Babesia villiformis.

5. The use of cordycepin according to claim 1 in the preparation of a medicament for treating Babesia infection, characterized in that: The drug is suitable for oral administration.

6. The use of cordycepin according to claims 1-5 in the preparation of a medicament for treating Babesia infection, characterized in that: The drug is formulated as any one of the following dosage forms: tablet, granule, capsule, oral liquid, injection, or suspension.

7. A pharmaceutical composition for treating Babesia infection, characterized in that: The composition comprises a therapeutically effective amount of cordycepin and a pharmaceutically acceptable carrier or excipient.

8. A pharmaceutical composition for treating Babesia infection according to claim 7, characterized in that: The cordycepin mentioned above has the molecular formula: .

9. A pharmaceutical composition for treating Babesia infection according to claim 7, characterized in that... According to any of the foregoing uses, the drug is used to reduce and / or delay host cell damage caused by Toxoplasma gondii infection.

10. Use of cordycepin in the preparation of drugs for the treatment of toxoplasmosis infection.

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