Use of pemetrexed in the preparation of anticoccidial drugs

Abstract

This invention relates to the field of biomedical technology and provides the application of pemetrexed in the preparation of anticoccidial drugs. This invention discloses a novel application of pemetrexed in the preparation of anticoccidial drugs, verifying that the compound has clear anti-parasitic activity in vitro, significantly improves chick survival rate, weight gain, reduces oocyst expulsion, and alleviates cecal lesions in vivo, with an anticoccidial index (ACI) rating of moderate efficacy. Furthermore, it exhibits low cytotoxicity and good safety, providing a new candidate and application direction for anticoccidial drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of pemetrexed in the preparation of anticoccidial drugs. Background Technology

[0002] Coccidiosis in chickens is a major disease in large-scale chicken farming. Among them, Eimeria tenella is the most pathogenic, mainly parasitizing the epithelial cells of the cecum in chickens. It can cause acute intestinal bleeding and inflammation in chickens, resulting in severe bloody feces and stunted growth. The mortality rate in acute infection can reach more than 50%, causing huge economic losses to large-scale chicken farming. At the same time, it can also lead to immunosuppression in chickens, increasing the risk of secondary infections, which seriously restricts the large-scale and intensive development of chicken farming.

[0003] Currently, the prevention and control of coccidiosis in chickens relies primarily on anticoccidial drugs, as vaccines cannot replace medication due to their limitations. Existing drugs are mainly divided into two categories: ionotropic antibiotics and chemically synthesized drugs. Long-term use of the former can disrupt the intestinal microecology and carries the risk of residues, while the latter suffers from problems such as single target, significant drug resistance, and insufficient safety. Overall, there is a lack of diverse drugs, and a shortage of highly effective and safe new drugs specifically targeting Eimeria tenella.

[0004] In the screening of anticoccidial drugs, traditional in vitro models have drawbacks such as poor accuracy, complex operation, and difficulty in standardization. However, the transgenic Eimeria tenella in vitro model can accurately simulate the key developmental stages of the coccidia in the host, and has the technical advantages of high throughput, high specificity, and good stability, making it an ideal tool for screening novel anticoccidial drugs.

[0005] Pemetrexed is a multi-target antifolate antitumor drug containing a pyrrolopyrimidine group. It blocks target cell proliferation by inhibiting the activity of key folate-dependent enzymes, interfering with folate metabolism and nucleic acid synthesis, and is currently used for the treatment of malignant tumors. To date, there are no reports on its anticoccal activity in chickens.

[0006] In summary, existing anticoccidial drugs for chickens have obvious shortcomings, and the development of new drugs is lagging behind. There is an urgent need to find new drugs to address the challenges facing the chicken farming industry. Summary of the Invention

[0007] The purpose of this invention is to provide the application of pemetrexed in the preparation of anticoccidial drugs, in order to solve the problems mentioned in the background art.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] On the one hand, the present invention provides the application of pemetrexed in the preparation of anticoccidial drugs.

[0010] On the other hand, the present invention also provides an anticoccidial drug, the drug comprising pemetrexed.

[0011] Compared with the prior art, the specific beneficial effects of the present invention are as follows:

[0012] This invention has determined the inhibitory effect of pemetrexed on Eimeria tenella at the micromolar level and systematically verified the in vivo anticoccidial effect of pemetrexed, filling the gap in related drugs in the prior art. Attached Figure Description

[0013] Figure 1 This refers to the initial screening results of the drug library provided in Embodiment 1 of the present invention;

[0014] Figure 2 The insect inhibition rate of pemetrexed (10 μM) provided in Example 1 of the present invention;

[0015] Figure 3 The in vitro antiparasitic effect of pemetrexed provided in Embodiment 1 of the present invention;

[0016] Figure 4 The results of the toxicity test of pemetrexed on MDBK cells and HepG2 cells provided in Example 2 of this invention;

[0017] Figure 5 The survival curve of the anticoccidial efficacy test provided in Example 3 of the present invention;

[0018] Figure 6 The relative weight gain rate in the anticoccidial efficacy test provided in Example 3 of this invention;

[0019] Figure 7 This is a representative cecal lesion from the anticoccidial efficacy test provided in Example 3 of the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0021] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0022] Example 1: Experimental analysis of the in vitro inhibitory effect of pemetrexed on Eimeria tenella:

[0023] A preliminary screening of 1770 existing compounds was conducted using a transgenic Eimeria tenella in vitro model. The specific experimental steps were as follows: Bovine kidney cells (MDBK) were seeded in 96-well black transparent bottom culture plates and cultured until the cell monolayer confluence reached more than 90%. Freshly decapitated sporozoites were then added at a rate of 1 × 10⁻⁶ per well. 5The cells were seeded at a density of [missing information] in cell culture plates and incubated at 41°C with 5% CO2 for 3 h. After that, the supernatant was aspirated, and each well was gently washed twice with sterile PBS. The cell state was observed under a microscope to confirm that no cells were detached. One test compound (concentration of 10 μM) was added to each well, and positive control wells and negative control wells were also set up. After the samples were added, the culture plate was gently shaken to mix the drug and the culture medium thoroughly. Then the culture plate was incubated at 41°C with 5% CO2 for another 42 h. After the samples were collected, the luciferase detection substrate and detection buffer were added according to the instructions and the detection was performed immediately using a multi-functional microplate reader.

[0024] For compounds with an insecticidal inhibition rate greater than 50% obtained in the initial screening, the insecticidal effect was further verified by single-agent verification (concentration of 10 μM), with each compound replicated in 2 wells.

[0025] As shown in Figure 1, during the screening of 1770 compounds for their insecticidal activity, 26 compounds with inhibition rates higher than 50% were initially identified, with pemetrexed being one of them; Figure 2 As shown, the anticoccal activity of pemetrexed was re-verified, and the results showed that its anti-insect rate at a concentration of 10 μM was 54.31% (mean), which was consistent with the results of the initial screening with an inhibition rate of more than 50%, and the verification results had good reproducibility.

[0026] To more comprehensively and quantitatively evaluate the in vitro anticoccidial activity of pemetrexed, a series of concentration gradients were set up for drug administration. The inhibitory effect of the drug on chicken coccidia at different concentrations was detected, dose-response curves were plotted, and the half-maximal effective concentration (EC50) was calculated. 50 This objectively reflects the insecticidal ability and efficacy of the compound. The specific experimental steps are as follows: Prepare the compound at the following concentration gradients: 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM. After preparation, mix thoroughly and set aside. Bovine kidney cells (MDBK) are seeded in 96-well black transparent bottom culture plates and cultured until the cell monolayer confluence reaches more than 90%. Freshly decapitated and counted sporozoites are seeded into the cell plate and incubated in a 41℃, 5% CO2 incubator for 3 h. After washing twice with sterile PBS, the cell state is observed under a microscope. Different concentrations of the prepared drug are added to the corresponding wells, with 3 replicates for each concentration. The cells are then incubated in a 41℃, 5% CO2 incubator for 42 h. After collecting the samples, the luciferase detection substrate and detection buffer are added according to the instructions and the detection is performed immediately using a multi-functional microplate reader.

[0027] like Figure 3 As shown, pemetrexed anticoccal activity EC 50 It is 9.65 μM.

[0028] Example 2: Detection of pemetrexed cytotoxicity to host cells:

[0029] The biocompatibility of a drug is an indispensable core indicator for evaluating its development and application potential, especially in the development of antiparasitic drugs. Candidate compounds must exhibit high levels of antiparasitic activity while demonstrating good safety to host cells and avoiding significant cytotoxicity. Based on this, this invention selected bovine kidney cells (MDBK) and human liver cancer cells (HepG2) as in vitro evaluation models to investigate the potential toxic effects of pemetrexed on host cells within a concentration gradient range of 0-1000 μM. Through in vitro cytotoxicity testing, the safe concentration range for use of this compound was determined.

[0030] The specific experimental steps were as follows: MDBK cells and HepG2 cells were digested with trypsin and counted. The cell density was adjusted with DMEM complete medium containing 10% FBS, and the cells were seeded into 96-well cell culture plates at 100 μL (1×10⁶ cells / well). 4 (100 cells), culture plates were placed in a 37℃, 5% CO2 incubator and cultured until the cell monolayer confluence reached 90% or more. Different concentrations of pemetrexed were added to each well, with separate control wells (0.1% DMSO solvent) and blank wells. Each concentration was tested in triplicate. MDBK cell culture plates were then incubated at 41℃, 5% CO2 for 42 h; HepG2 cell culture plates were incubated at 37℃, 5% CO2 for 42 h. After incubation, the drugs in the wells were aspirated, and each well was gently washed three times with sterile PBS. Then, 100 μL of serum-free DMEM medium and 10 μL of MTS reagent were added to each well, gently vortexed to mix, and the culture plates were returned to the incubator for 1.5 h in the dark. Detection was performed using a multi-functional microplate reader.

[0031] Cytotoxicity assay results showed that in MDBK cells, the half-maximal toxic concentration (TC) of pemetrexed was [value missing]. 50 ) higher than 1000μM (e.g. Figure 4 (As shown); In HepG2 cells, pemetrexed TC 50 Also greater than 1000 μM (e.g.) Figure 4 As shown); accordingly, the embodiments of the present invention clarify the safety interval (SI) of pemetrexed: SI > 103.6, i.e., TC 50 With EC 50 The ratio of SI to the drug's antiparasitic activity is as follows: the higher the SI value, the stronger the drug's antiparasitic ability and the lower its cytotoxicity.

[0032] Example 3: Experimental analysis of the in vivo anticoccidial effect of pemetrexed against Eimeria tenella:

[0033] To further verify the anticoccidial activity of pemetrexed and to translate in vitro experimental results into in vivo application references, clarifying its anticoccidial effect, appropriate dosage, and potential application value in live animal models, and to overcome the limitations of in vitro experiments in simulating the physiological environment of the body, an in vivo anticoccidial experiment of pemetrexed was conducted based on the completion of in vitro antiparasitic efficacy determination. This invention uses the anticoccidial index (ACI) score as the core evaluation indicator. The ACI value was calculated by measuring the survival rate, relative weight gain rate, fecal oocyst count, and lesion score of the experimental animals, thus systematically evaluating the actual antiparasitic efficacy of the drug in vivo. The specific experimental steps are as follows: Twenty-four 3-day-old chicks were acclimatized for 2 days and then randomly divided into 4 groups of 6 chicks each. They were weighed and marked. The group design was as follows: non-infected group; positive control group (dicrazine group); infected model group; pemetrexed group; the transgenic Eimeria tenella oocysts used for infection were sporulated oocysts passaged and preserved in our laboratory. Before infection, the oocyst suspension was centrifuged at 3500 rpm for 6 seconds. After 1 minute, the supernatant was discarded, and the precipitate was resuspended in sterile PBS and washed three times to remove potassium dichromate. Except for the non-infected group, each chick in the other groups was orally administered 1.5 × 10⁻⁶ ppm. 4 One oocyst was identified. Oral administration began on the second day after infection, once daily for 7 consecutive days. The positive control group received diclazuril, with free access to the drinking water, and the solution was changed daily. The infected model group received an equal volume of the solution (0.5% DMSO + 40% PEG300 + 5% Tween-80 + 54.5% saline) via gavage at the same time as the treatment group. The uninfected group received no treatment. During the treatment period, the weight of each chicken was measured, and mortality, mental status, and food and water intake were recorded daily. Fresh feces were collected from each group on the 8th day after coccidiosis infection, clearly labeled, and used for oocyst counting. The collected fecal samples were processed as follows: three 1g fecal samples were weighed from each group, and 9 mL of saturated physiological saline was added and mixed thoroughly to prepare a 10-fold diluted fecal suspension. The total number of oocysts in four large squares was counted using a counting chamber. The mean oocyst count (N) of the three samples in each group was calculated. The oocyst count per gram of feces (OPG) = (C × D) / W; the concentration of the oocyst suspension: C oocysts / mL; D is the total volume of the suspension (mL); W is the fecal mass (g); C = (N / 4) × 10 × 10 4 = (N / 4) × 10 5 Substituting the above conditions: OPG = [(N / 4) × 10 5 OPG = N × 2.5 × 10⁻¹⁰ g / 1 g 5 ;

[0034] The ovum value was calculated based on the ovum ratio, as shown in Table 1:

[0035] Oocyte ratio = (Number of ovarian follicles in the treatment group or the non-infected / non-treatment group) / (Number of ovarian follicles in the infected / non-treatment group) × 100%

[0036] Table 1

[0037]

[0038] After the administration was completed, all surviving chicks were euthanized by cervical dislocation, and the bilateral cecums were immediately removed for lesion scoring.

[0039] like Figure 5 As shown, no chicks in the pemetrexed group died during the experimental period, and their survival rate was significantly better than that of the infected control group. The difference in survival status between the two groups was significant, suggesting that the drug can effectively improve chick mortality caused by coccidiosis infection and has a significant protective effect on infected chicks.

[0040] like Figure 6 As shown, throughout the entire experimental period, the relative weight gain rate of chicks in the pemetrexed group was significantly higher than that in the infected control group. This result indicates that pemetrexed can effectively alleviate the inhibitory effect of coccidiosis infection on the growth and development of chicks, further confirming its positive anti-coccidiosis effect in vivo.

[0041] As shown in Table 2, the fecal oocyst count in the pemetrexed group was significantly lower than that in the infected control group. This result indicates that pemetrexed can effectively inhibit the reproduction and oocyst excretion of coccidia in chicks, reduce the infection load, and significantly alleviate the severity of coccidia infection. This further verifies its in vivo anticoccidia activity and provides direct in vivo experimental evidence for the anticoccidia effect of this drug.

[0042] Table 2. Oocyst values ​​in the anticoccidial efficacy test;

[0043]

[0044] like Figure 7 As shown, the cecal lesions in the pemetrexed group of chicks were less severe than those in the infected control group. Typical pathological damage such as cecal mucosal congestion, hemorrhage and swelling were alleviated to some extent, indicating that the drug has a certain protective effect against cecal damage caused by coccidiosis.

[0045] Based on a comprehensive evaluation of factors including chick survival, weight gain, oocyst excretion, and improvement in cecal lesions, the chicks were scored according to the anticoccidial index (ACI) calculation standard. The results are shown in Table 3.

[0046] Table 3

[0047]

[0048] It can be seen that pemetrexed exhibits a moderate level of anticoccidial effect in vivo, effectively improving the survival rate of infected chicks, alleviating growth inhibition, reducing oocyst expulsion, and reducing cecal tissue damage. Under the same experimental conditions, the positive control drug diclazuril showed a highly effective anticoccidial effect, with a more prominent overall therapeutic effect. The above results fully confirm that pemetrexed has a clear and stable anticoccidial activity in vivo.

[0049] In summary, the embodiments of this invention have demonstrated that pemetrexed exhibits clear anticoccal activity in vitro, inhibiting coccidia proliferation in a dose-dependent manner and yielding stable EC. 50 The value provides reliable in vitro experimental evidence for the anti-coccidia effect; in vivo, it can significantly improve the survival rate of coccidia-infected chicks, improve their growth performance and relative weight gain rate, reduce the amount of oocysts excreted, and alleviate cecal tissue lesions and damage.

[0050] Based on the comprehensive evaluation of the anticoccidial index (ACI), pemetrexed showed a moderate level of anticoccidial efficacy and had a clear and stable in vivo anticoccidial activity. This drug can provide a new candidate compound for the existing anticoccidial drug system, broaden the selection range of anticoccidial drugs, and has the potential to be further developed into a novel anticoccidial formulation.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of pemetrexed in the preparation of anticoccidial drugs.

2. The application of pemetrexed according to claim 1 in the preparation of anticoccidial drugs, characterized in that, The anticoccidial drug targets the species *Eimeria tenella*.

3. An anticoccidial drug, characterized in that, The medication includes pemetrexed.

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