Application of docetaxel in preparation of drugs for resisting toxoplasmosis

Abstract

The invention discloses an application of docetaxel in preparation of drugs for resisting toxoplasmosis, and belongs to the technical field of medicines. Through a series of experimental studies, it is found that docetaxel can significantly delay the onset time of mice infected with toxoplasma gondii virulent strains and effectively reduce the death rate of mice infected with toxoplasma gondii attenuated strains. Docetaxel shows a relatively strong inhibition effect in the aspect of treating related diseases caused by a virulent strain or a low virulent strain of toxoplasma gondii, and can effectively slow down the infection process and prolong the life cycle of a host. In combination with in-vitro cell experiment and in-vivo animal experiment results, the taxane drug has a good treatment prospect, can be used as a development basis of a novel toxoplasmosis-resistant drug, and promotes the realization of a novel treatment scheme.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of docetaxel in the preparation of anti-toxoplasmosis drugs. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Toxoplasmosis is caused by Toxoplasma gondii (… Toxoplasma gondii Toxoplasmosis is a widespread zoonotic parasitic disease caused by Toxoplasma gondii, infecting almost all warm-blooded animals and posing a serious threat to human health and livestock development. In individuals with normal immune function, Toxoplasma gondii infection often presents as a latent infection. However, for immunocompromised or immunosuppressed individuals (such as AIDS patients, organ transplant recipients, and patients with malignant tumors) and pregnant women, Toxoplasmosis infection can lead to fatal consequences, such as miscarriage, fetal malformations, intellectual disability, and stillbirth. Currently, clinical treatment of toxoplasmosis mainly relies on traditional chemical drugs, such as the combination of sulfadiazine and pyrimethamine, spiramycin, and azithromycin. These drugs mainly exert their effects by inhibiting the parasite's folic acid metabolism or interfering with its proliferation process, but they generally have limitations: on the one hand, they mainly target the trophozoite stage of Toxoplasma gondii and are not very effective against tissue cysts, making it difficult to completely eliminate the parasite, and relapse is common after drug withdrawal; on the other hand, these drugs are often accompanied by significant side effects such as bone marrow suppression, blood toxicity, and allergic reactions, and long-term use may induce drug resistance in the parasite strain. In addition, existing drugs have limited efficiency in penetrating cell barriers and targeting intracellular parasites, and their therapeutic effects are particularly unsatisfactory for infections in the central nervous system and the eyes.

[0004] In recent years, researchers have explored new strategies such as extracts from traditional Chinese medicine (e.g., artemisinin, matrine) and nano-drug delivery systems to improve drug targeting and efficacy. However, to date, there is still a lack of ideal drugs that can effectively inhibit intracellular Toxoplasma gondii proliferation, block the spread of the parasite between host cells, and effectively delay disease progression. Therefore, the development of novel anti-toxoplasmosis drugs, especially compounds that can effectively inhibit intracellular parasite proliferation, prevent plaque formation, delay disease progression, and improve host survival, has become an urgent need in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of docetaxel in the preparation of anti-toxoplasmosis drugs. Through a series of experimental studies, this invention has found that docetaxel can significantly delay the onset of disease in mice infected with virulent strains of Toxoplasma gondii and effectively reduce the mortality rate in mice infected with attenuated strains. Docetaxel exhibits a strong inhibitory effect in treating diseases caused by virulent or attenuated strains of Toxoplasma gondii, effectively slowing the infection process and prolonging the host's survival. Combining the results of in vitro cell experiments and in vivo animal experiments, taxane drugs show promising therapeutic potential and can serve as the basis for the development of novel anti-toxoplasmosis drugs, promoting the realization of new treatment regimens.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: A first aspect of the invention provides the use of docetaxel in the preparation of anti-toxoplasmosis drugs.

[0007] Docetaxel (CAS: 114977-28-5) is a microtubule depolymerization inhibitor with an IC50 value of 100%. 50 The value was 0.2 μM. Docetaxel is a semi-synthetic analog of paclitaxel that attenuates the expression of bcl-2 and bcl-xL genes. Docetaxel arrests the G2 / M cell cycle, leading to apoptosis. Docetaxel has antitumor activity. However, there are currently no studies using docetaxel to treat toxoplasmosis.

[0008]

[0009] Through a series of experimental studies, this invention has found that docetaxel can significantly delay the onset of disease in mice infected with virulent strains of Toxoplasma gondii and effectively reduce the mortality rate in mice infected with attenuated strains. Docetaxel exhibits a strong inhibitory effect in treating diseases caused by virulent or attenuated strains of Toxoplasma gondii, effectively slowing the infection process and prolonging host survival. Combining the results of in vitro cell experiments and in vivo animal experiments, docetaxel shows promising therapeutic potential and can serve as a basis for the development of novel anti-toxoplasmosis drugs, promoting the realization of new treatment regimens.

[0010] In some embodiments of the present invention, the toxoplasmosis includes toxoplasmosis caused by infection with the virulent strain RH or the attenuated strain ME49 of Toxoplasma gondii.

[0011] In some embodiments of the present invention, the toxoplasmosis includes toxoplasmosis caused by Toxoplasma gondii infection of human cervical cancer cells (HeLa).

[0012] In some embodiments of the present invention, the anti-Toxoplasma gondii drug includes a drug that inhibits the proliferation of Toxoplasma gondii within cells.

[0013] Preferably, the cells include human cervical cancer cells.

[0014] In some embodiments of the present invention, the anti-Toxoplasma gondii drug includes a drug that inhibits the formation of Toxoplasma gondii plaques.

[0015] In some embodiments of the present invention, the anti-toxoplasmosis drug includes drugs that delay the progression of toxoplasmosis and / or improve host survival.

[0016] In some embodiments of the present invention, the dosage of the anti-toxoplasmosis drug, calculated based on the mouse dosage, is 25 mg / kg once every three days.

[0017] In some embodiments of the present invention, the dosage form of the anti-toxoplasmosis drug includes tablets, capsules, granules, drops, pills, lyophilized products, granules, ointments, or injections.

[0018] In some embodiments of the present invention, the anti-toxoplasmosis drug is administered orally, by gavage, or by subcutaneous injection.

[0019] In some embodiments of the present invention, the anti-toxoplasmosis drug is administered to humans or non-human mammals. The non-human mammals mentioned in this invention may include mice, rats, rabbits, dogs, etc.

[0020] In some embodiments of the present invention, the active ingredient in the anti-toxoplasmosis drug is docetaxel or its pharmaceutically acceptable salts, hydrates, or solvates.

[0021] The pharmaceutically acceptable salt described in this invention refers to a salt formed by docetaxel and a non-toxic inorganic or organic acid. The inorganic acid can be hydrochloric acid, sulfuric acid, phosphoric acid, etc.; the organic acid can be acetic acid, propionic acid, lactic acid, oxalic acid, maleic acid, fumaric acid, malic acid, etc.

[0022] In some embodiments of the present invention, the anti-toxoplasmosis drug further includes pharmaceutically acceptable excipients.

[0023] The pharmaceutically acceptable excipients described in this invention refer to excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, including but not limited to: binders, fillers, disintegrants, pH adjusters, surfactants, adjuvants, diluents, preservatives, etc.

[0024] The beneficial effects of this invention are as follows: This invention discloses the use of docetaxel in the preparation of an anti-toxoplasmosis drug. The drug exhibits low toxicity in in vitro cell experiments, particularly showing no significant toxicity to human cervical cancer cells at concentrations below 6.25 nM, indicating potentially good safety for clinical use. Docetaxel demonstrates potent inhibitory activity against Toxoplasma gondii RH-GFP strain, with a half-maximal inhibitory concentration (EC50) of [missing value]. 50 The concentration of docetaxel was 2.21 nM, demonstrating that the drug possesses highly effective anti-Toxoplasma gondii activity and can significantly inhibit the proliferation and spread of Toxoplasma gondii. In in vitro plaque experiments, docetaxel significantly inhibited plaque formation in Toxoplasma gondii RH strains, further validating its inhibitory effect on Toxoplasma gondii. More importantly, in vivo mouse experiments showed that docetaxel not only significantly delayed the onset of disease in mice infected with virulent Toxoplasma gondii RH strains but also effectively improved the survival rate of mice infected with attenuated Toxoplasma gondii ME49 strains, demonstrating excellent therapeutic potential. Furthermore, docetaxel also provides a new potential option for treating Toxoplasma gondii infections resistant to traditional drugs (such as sulfonamides combined with pyrimethamine). Therefore, the docetaxel provided by this invention has clinical application prospects in the treatment of toxoplasmosis and can provide new ideas and effective treatment methods for the development of anti-toxoplasmosis drugs. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 This is the cytotoxicity curve of taxane drugs against HeLa in Example 1 of the present invention.

[0027] Figure 2 This is the inhibition curve of taxane drugs against HeLa intracellular Toxoplasma gondii in Example 2 of the present invention.

[0028] Figure 3 This describes the effect of taxane drugs on plaque formation in Toxoplasma gondii RH strain in Example 3 of the present invention.

[0029] Figure 4 This is the survival curve of mice infected with a highly virulent strain of Toxoplasma RH using taxane drugs in Example 4 of this invention.

[0030] Figure 5 This is the survival curve of mice infected with attenuated strain ME49 Toxoplasma gondii using taxane drugs in Example 4 of the present invention. Detailed Implementation

[0031] This invention provides the application of docetaxel in the preparation of anti-toxoplasmosis drugs. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention. Docetaxel is a taxane drug used as an antitumor, photosensitizer, and antimalarial agent, but its role in toxoplasmosis is unclear. The test materials used in this invention are all common commercial products and can be purchased on the market.

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0033] Example 1: Cytotoxicity assay of docetaxel 1. Material Description Human cervical cancer cells (HeLa), docetaxel (MedChemExpress), CCK-8 kit (Beyotime), dimethyl sulfoxide (DMSO) (Solepro), 96-well cell culture plate (Konning), DMEM medium (Gibco), and fetal bovine serum (FBS) (Gibco).

[0034] 2. Experimental Methods HeLa cells were used at a concentration of 2-5 × 10⁻⁶ cells per well. 3 Cells were seeded at a density of 1000 mcg / well in 96-well microplates and incubated for 24 h. Taxanes were then added to final concentrations of 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 nM. After 48 h of incubation, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for another 1–2 h. The absorbance (OD) was then measured at 450 nm using a microplate reader. Blank wells (without taxanes), background wells (culture medium only), and sample wells (containing different concentrations of taxanes) were included. Cell inhibition rate was calculated using the following formula: Inhibition rate (%) = 1 - (OD) 样品 -OD 背景 ) / (OD 空白 -OD 背景 ×100%. At least three replicates were performed for each concentration. The dose-response curve was fitted using nonlinear regression with data processing and graphing software, and the half-maximal inhibitory concentration (IC50) was calculated. 50 ).

[0035] See results Figure 1 When the concentration of taxanes was below 6.25 nM, they did not show significant toxicity to the growth of HeLa cells; the half-maximal inhibitory concentration (IC50) for HeLa cell proliferation was calculated. 50 The value is 123 nM.

[0036] Example 2: Docetaxel's inhibitory effect on intracellular Toxoplasma gondii 1. Material Description Human cervical cancer cells (HeLa), docetaxel (MedChemExpress), RH-GFP strain (donated by Shanxi Agricultural University), 24-well cell culture plate (Kangning), DMEM medium (Gibco), and fetal bovine serum (FBS) (Gibco).

[0037] 2. Experimental Methods After culturing host cells (HeLa) to 90% confluence, RH-GFP strains were inoculated at an MOI of 3, and uninvaded Toxoplasma gondii were removed after 3 h. Subsequently, different concentrations of taxane compounds were added to the drug-treated groups; the control group received an equal volume of solvent (0.1% DMSO), and both groups were cultured under the same conditions for 24 h. Images were then acquired using a fluorescence microscope (Carl Zeiss AG, Germany) to observe the fluorescence signal of Toxoplasma gondii within the cells. The obtained images were quantitatively analyzed using ImageJ software, and the relative proliferation level of Toxoplasma gondii in each treatment group was reflected by measuring the fluorescence area. All experiments were independently repeated three times, and results are expressed as mean ± standard deviation (SD). Dose-response curves were fitted using nonlinear regression and CC50 values ​​were calculated using data processing and graphics software.

[0038] See results Figure 2 The half-maximal inhibitory concentration (IC50) of docetaxel against Toxoplasma gondii RH-GFP tachyzoites. 50 The value is 2.21 nM.

[0039] Example 3: Inhibition experiment of docetaxel on Toxoplasma gondii plaque formation 1. Material Description Human foreskin fibroblasts (HFF), docetaxel (MedChemExpress), RH strain (donated by Shanxi Agricultural University), 12-well cell culture plate (Kangning), DMEM medium (Gibco), fetal bovine serum (FBS) (Gibco), dimethyl sulfoxide (DMSO) (Beyotime), 4% paraformaldehyde (Sewell), and 0.1% crystal violet solution (Solepro).

[0040] 2. Experimental Methods Purified RH tachyzoites were seeded at 100 per well into HFF cells to establish an in vitro infection model. The drug-treated groups received 5 nM docetaxel, while the control group received 0.1% DMSO. Cells were cultured under the same conditions for 7 days. Subsequently, host cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. After staining, the culture plates were scanned, and the number and average area of ​​plaques were compared between the different treatment groups to assess the effect of the drug on Toxoplasma gondii plaque formation.

[0041] See results Figure 3 Docetaxel has a significant inhibitory effect on the formation of Toxoplasma gondii RH tachyzoite plaques.

[0042] Example 4: Docetaxel exhibits in vivo anti-Toxoplasma gondii activity. 1. Material Description C57BL / 6 mice, RH and ME49 strains (donated by Shanxi Agricultural University), docetaxel (MedChemExpress), Tween-80 (Solepro), PEG-300 (Solepro), physiological saline (Beyotime), DMSO (Beyotime). Solvent preparation: Mix 10% DMSO, 40% PEG300, 5% Tween-80 and physiological saline in sequence, and make sure to mix well before adding the next portion of solvent.

[0043] 2. Experimental Methods To evaluate the protective effect of docetaxel in an in vivo Toxoplasma gondii infection model, healthy mice were randomly assigned to groups for the experiment. Mice in the experimental group were intraperitoneally injected with either Toxoplasma gondii RH (a virulent strain) or ME49 (a weak strain) to establish an infection model, while the control group received an equal volume of sterile PBS. Drug administration began 24 hours after infection, and infected mice were randomly divided into two groups: a solvent control group (treated with 10% DMSO, 40% PEG-300, 5% Tween 80, and physiological saline) and a taxane treatment group (dose 25 mg / kg, prepared using the same solvent). The drug was administered intraperitoneally every 3 days for a total of 3 treatments. During the experiment, the mice's mental state, activity level, and survival rate were observed daily at regular intervals (7:00 AM, 12:00 PM, and 7:00 PM). All animal experiments were conducted in accordance with animal ethics guidelines and approved by the relevant ethics committee.

[0044] See results Figure 3 and Figure 4 .like Figure 3 As shown, the mortality rate of mice in the solvent + 100RH group reached 70% on day 9 post-infection, while mice treated with docetaxel did not die within the same timeframe, indicating that the drug can significantly delay the disease progression in mice infected with virulent Toxoplasma gondii strains. Figure 4As shown, in the experiment of infecting the attenuated strain ME49, all mice in the solvent + 500mg ME49 group died on day 23 post-infection, while approximately 40% of the mice in the docetaxel + 500mg ME49 treatment group survived, suggesting that docetaxel can effectively improve the survival rate of infected mice. In conclusion, docetaxel has a significant protective effect against Toxoplasma gondii infection in mice.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of docetaxel in the preparation of an anti-toxoplasmosis drug.

2. Use according to claim 1, wherein The toxoplasmosis includes toxoplasmosis caused by infection of human cervical cancer cells with virulent RH or attenuated ME49 strain of Toxoplasma gondii.

3. The use according to claim 1, wherein The toxoplasmosis includes toxoplasmosis caused by infection of human cervical cancer cells with virulent RH or attenuated ME49 strain of Toxoplasma gondii.

4. The use according to claim 1, wherein The anti-toxoplasma drug includes a drug that inhibits the proliferation of intracellular Toxoplasma gondii. Preferably, the cells include human cervical cancer cells.

5. The use according to claim 1, wherein The anti-toxoplasma drug includes a drug that inhibits the formation of Toxoplasma gondii plaques.

6. The use according to claim 1, wherein The anti-toxoplasma drug includes a drug that delays the progression of toxoplasmosis and / or increases the survival rate of the host.

7. The use according to claim 1, wherein The dosage of the anti-toxoplasma drug is 25 mg / kg once every three days in mice.

8. The use according to claim 1, wherein The dosage form of the anti-toxoplasma drug includes tablets, capsules, powders, drops, pills, lyophilized products, granules, ointments, or injections.

9. The use according to claim 1, wherein The administration of the anti-toxoplasma drug is oral, intragastric, or subcutaneous injection.

10. The use according to claim 1, wherein The administration of the anti-toxoplasma drug is oral, intragastric, or subcutaneous injection. The administration of the anti-toxoplasma drug is oral, intragastric, or subcutaneous injection.

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