Application of maduramicin ammonium in preparation of medicine for treating helminthosis
By using the method of madumycin ammonium in DMSO preparation, the problem of poor treatment of existing drugs on Echinococcus and trichinidiae was solved, and efficient killing of worms was achieved, especially the broad-spectrum killing effect on the protocelebrates and trichinidiae of Echinococcus.
Patent Information
- Application Number
- CN202510643400.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-19
- Publication Date
- 2025-08-22
AI Technical Summary
Existing drugs such as albendazole and mebendazole can only inhibit echinococcosis, but cannot completely kill echinococcosis. It has limited effect on trichinidae muscle cysts, resulting in a low cure rate and side effects. It is necessary to develop safe and efficient helminthic disease treatment drugs.
Madumycin ammonium is dissolved in DMSO to prepare the stock solution, which is used in worm culture medium. By interfering with the mitochondrial membrane potential, it induces apoptosis cascade, and achieves efficient killing of the protocelebone of the multi-apart echinococcal genus and trichinidae.
In vitro experiments, the mortality rate of madunomycin ammonium on the protocerebrosus echinococcosis reached 100%, and the killing rates of trichinidae muscle larvae, adult and neonatal larvae were 93.87%, 93.62% and 99.35%, respectively, showing broad-spectrum insecticidal properties and having good tissue penetration ability.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of development and application of antiparasitic drugs, and relates to the application of maduramicin ammonium, and particularly to the application of maduramicin ammonium in the preparation of drugs for treating helminthiasis. Background Art
[0002] Helminths are multicellular invertebrates that rely on muscle contractions for peristaltic locomotion. They primarily include various animals belonging to the phyla Platyhelminthes, Annelida, Nematoda, and Acanthocephala. Helminthiasis is a disease caused by parasitic worms that infect humans or animals, including Echinococcosis and trichinosis.
[0003] Alveolar echinococcosis (AE), also known as alveolar echinococcosis, is a major zoonotic parasitic disease caused by the larvae of the tapeworm Echinococcus multilocularis (Em), a member of the family Taeniadae, class Platyhelminthes, parasitizing the liver, lungs, and other tissues of humans and animals. It is predominantly prevalent in livestock-rich regions of the Northern Hemisphere. Globally, 18,000 new cases of AE occur annually, resulting in approximately 666,000 DALYs. Echinococcus multilocularis larvae asexually reproduce in organs such as the liver of humans or other intermediate hosts, leading to space-occupying lesions, organ dysfunction, and even death.
[0004] Trichinella spp. are important zoonotic parasites, causing trichinellosis (Trich), a global threat to public health and food safety. The genus comprises 13 recognized species and genotypes, of which T. spiralis (infecting domestic pigs and rodents) and T. britovi (adapted to wildlife hosts) are the primary pathogenic species. Infection occurs through ingestion of raw or undercooked meat containing live larval cysts. The larvae develop into adults in the small intestine and produce neonates, which circulate and invade skeletal muscle, forming cysts and causing fever, myalgia, eosinophilia, and, in severe cases, myocarditis or encephalitis.
[0005] Currently, the primary global treatment for multilocular echinococcosis and trichinosis is oral albendazole (ABZ) and mebendazole. However, studies have shown that these two drugs only inhibit the parasitism of multilocular echinococcosis but cannot completely kill the cysts, resulting in low cure rates and requiring long-term use, which can lead to side effects such as hepatotoxicity. Targeting Trichinella, these two drugs only kill intestinal adult worms and migratory larvae, but have limited effectiveness against muscle cysts, making them suitable only for patients in the early stages of infection. Therefore, the development of safe, effective, and cost-effective drugs for the treatment of AE and Trichinosis is of great significance.
[0006] Maduramicin ammonium is a polyether ionophore antibiotic primarily used to control coccidiosis, a primary parasitic infection in poultry, particularly chickens. It selectively binds to sodium and potassium ions on the coccidian cell membrane, disrupting the parasite's ion balance, leading to abnormal cytoplasmic osmotic pressure and mitochondrial dysfunction, thereby effectively killing coccidian trophozoites and schizonts. However, studies have shown that maduramicin ammonium has high biotoxicity, and overdose can cause poisoning or even death. Currently, there are no reports of its use in cross-species treatment of helminthic infections. Summary of the Invention
[0007] Based on the above, the object of the present invention is to provide the use of maduramicin ammonium in the preparation of a medicament for treating helminthiasis.
[0008] In the present invention, the helminthiasis is tapeworm disease or nematode disease.
[0009] Furthermore, the tapeworm is Echinococcus multilocularis, and the nematode is Trichinella spiralis.
[0010] Furthermore, the multilocular echinococcosis is a multilocular echinococcosis protoscolex.
[0011] The Trichinella spiralis is a Trichinella spiralis muscle larvae, an adult or a newborn larvae.
[0012] The present invention also provides a method for killing helminthiasis in vitro, which comprises dissolving maduramicin ammonium in DMSO to prepare a DMSO stock solution, and then adding the DMSO stock solution into the culture medium of the worms.
[0013] As a preferred embodiment of the technical solution of the present invention, the concentration of the DMSO stock solution is 10 mM.
[0014] Preferably, the worm culture conditions are 37° C. and 5% CO 2 . The worm is a tapeworm or a nematode, and further, the tapeworm is Echinococcus multilocularis and the nematode is Trichinella spiralis.
[0015] The beneficial effects of the present invention are:
[0016] The results of an in vitro efficacy test of maduramicin ammonium on the protoscolex of Echinococcus multilocularis show that the killing effect of maduramicin ammonium on the protoscolex is concentration- and time-dependent. At a concentration of 20 μM after 24 hours, the mortality rate of the protoscolex can reach 98.24%, and at a concentration of 50 μM after 24 hours, the mortality rate of the protoscolex can directly reach 100%. Meanwhile, the results of an in vitro efficacy test of maduramicin ammonium on Trichinella spiralis at different developmental stages show that the killing rate of maduramicin ammonium on Trichinella spiralis muscle larvae is 67.83% at a concentration of 50 μM after 72 hours, and reaches 93.87% at a concentration of 100 μM after 72 hours. The killing rates of maduramicin ammonium on Trichinella spiralis adults and newborn larvae are 93.62% and 99.35% respectively at a concentration of 50 μM after 24 hours, and reach 100% at a concentration of 100 μM after 24 hours, indicating a significant killing effect. This broad-spectrum insecticidal property across developmental stages may be due to its interference with mitochondrial membrane potential, which induces the opening of mitochondrial permeability transition pore and triggers a cell apoptosis cascade.
[0017] From a pharmacokinetic perspective, the lipophilicity of maduramicin ammonium gives it excellent tissue penetration, which is particularly important for the treatment of parasitic infections that require penetration through the cyst wall or muscle tissue to exert its efficacy. Furthermore, reports indicate that the drug has a metabolic half-life of 20-39 hours in the host, which is beneficial for maintaining effective therapeutic concentrations (Sharma N, Bhalla A, Varma S, Jain S, Singh S. Toxicity of maduramicin. Emerg Med J. 2005, 22(12):880-882).
[0018] In summary, the anti-helminthiasis therapeutic effect demonstrated by maduramicin ammonium has expanded the application range of this drug. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Effects of maduramicin ammonium on the protoscoleces of Echinococcus multilocularis in vitro (200×): A. Protoscoleces were incubated with DMEM containing DMSO, different concentrations of ABZ, and maduramicin ammonium (5-100 μM) for 24 h, and their viability was determined using 0.1% methylene blue solution; B. Morphology of protoscoleces in the blank group, DMSO negative control group, and ABZ positive control group; C. Morphology of protoscoleces incubated with maduramicin ammonium for 24 h; D. Morphology of protoscoleces incubated with maduramicin ammonium for 24 h after staining with 0.1% methylene blue;
[0020] Figure 2 Effects of different exposure times of maduramicin ammonium on the morphology of Trichinella spiralis muscle larvae in vitro (100×): A. The lethality of muscle larvae exposed to different concentrations of maduramicin ammonium for different exposure times in vitro; B. The morphology of muscle larvae in the blank group and the DMSO group; C. The morphology of dead larvae after exposure to maduramicin ammonium;
[0021] Figure 3 Effects of maduramicin ammonium on adult Trichinella spiralis at different exposure times in vitro (50×): A. The lethality of adult worms at different exposure times at different concentrations of maduramicin ammonium; B. The morphology of worms in the blank group and the DMSO group; C. The morphology of the dead worms after exposure to maduramicin ammonium.
[0022] Figure 4 Effects of maduramicin ammonium on newborn larvae of Trichinella spiralis at different exposure times in vitro (200×): A. The lethality of newborn larvae at different exposure times at different concentrations of maduramicin ammonium; B. Morphology of newborn larvae in the blank group and the DMSO group; C. Morphology of dead larvae after exposure to maduramicin ammonium.
[0023] Figure 5 These are the results of the toxicity experiment of maduramicin ammonium on mouse hepatic stellate cells. DETAILED DESCRIPTION
[0024] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
[0025] 1. Materials and Methods
[0026] 1.1 Biomaterials
[0027] The Echinococcus multilocularis strain used in this invention was derived from naturally infected plateau pikas in Yushu City, Qinghai Province, China, and the Trichinella spiralis strain used was derived from Henan Province, China. Both strains were maintained and propagated in the specialized laboratory of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The Echinococcus multilocularis protoscolex used in this invention was collected from Balb / c mice maintained and propagated in the animal laboratory of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The muscle larvae, adults, and neonatal larvae of Trichinella spiralis were collected from Kunming mice maintained and propagated in the animal laboratory of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.
[0028] 1.2 Main reagents and instruments
[0029] Reagents: Dulbecco's medium (DMEM), Dulbecco's alkaline phosphate solution (DPBS), fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Gibco, USA; 0.1% methylene blue staining solution was purchased from Beijing Solebow Technology Co., Ltd.; hydrochloric acid was purchased from Sinopharm Chemical Reagent Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Sigma, USA; ABZ and maduramicin ammonium were purchased from MedChemExpress, USA.
[0030] Instruments: Precision electronic balance was purchased from Merck Millipore, Germany; clean bench was purchased from Jiangsu Sujing Antai Company; constant temperature CO2 incubator was purchased from Thermo Fisher Scientific, USA; inverted fluorescence microscope was purchased from Zeiss, Germany; magnetic stirrer was purchased from Shanghai Meiyingpu Instrument Manufacturing Co., Ltd.; cell counter was purchased from Keqiao Medical Equipment Factory.
[0031] 1.3 The killing effect of maduramicin ammonium on the protoscolex of Echinococcus multilocularis
[0032] The protoscoleces of Echinococcus multilocularis were aseptically isolated from the hydatid cysts of Balb / c mice maintained by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. The protoscoleces were washed and precipitated five times with a DPBS solution (1%, 100 U / mL penicillin-streptomycin) to obtain clean, translucent protoscoleces. In a 96-well cell culture plate, 100 viable and morphologically intact protoscoleces were cultured in 200 μL of DMEM complete medium (10% FBS) and incubated at 37°C, 5% CO2 for 12 hours before drug addition. All drugs were prepared as 10 mM DMSO stock solutions and added to the protoscolecene culture medium at final concentrations of 50, 80, and 100 μM. Preliminary screening was performed for 24, 48, and 72 hours, respectively. Maduramicin ammonium was found to have a better killing effect, which was further refined. The killing effects of maduramicin ammonium and ABZ on Echinococcus multilocularis protoscolex were examined at final concentrations of 5, 10, 20, 50, 80, and 100 μM, respectively, at 6, 12, and 24 hours of incubation. Blank and 0.1% DMSO groups served as negative controls, while a 50 μM ABZ group served as a positive control. The effects of the drugs on the morphology and structural integrity of the protoscolex were observed under a microscope, and their viability was assessed by staining with 0.1% methylene blue. Protoscolex were mixed with 0.25% (v / v) methylene blue for 3 minutes, washed multiple times with DPBS, and observed under a microscope. Protoscolex staining blue indicated death. Protoscolex mortality was calculated as the mean number of dead protoscolex / mean total number of protoscolex × 100%. Each experiment was repeated three times for each drug concentration.
[0033] 1.4 The killing effect of maduramicin ammonium on Trichinella spiralis muscle larvae, adults and newborn larvae
[0034] Kunming mice used for seed preservation were killed by cervical dislocation, and the epidermis, viscera, and other organs were discarded, retaining only the muscle and bones. Muscle larvae were obtained by pepsin digestion. After counting, 1,000 muscle larvae were orally fed to healthy Kunming mice. To obtain adult worms, Kunming mice inoculated with 3-day-old muscle larvae were killed, and the small intestine was isolated and dissected longitudinally. The intestinal segments were cleaned with sterile saline and then spread flat on a gauze-covered plate. After incubation in sterile saline for 4 hours, the adults were collected. To obtain newborn larvae, Kunming mice inoculated with 5-day-old muscle larvae were killed, and adult worms were obtained. After washing five times with DPBS (1%, 100 U / mL penicillin-streptomycin), they were placed in a cell culture flask containing 10 mL of DMEM complete medium and incubated at 37°C, 5% CO2 for 24 hours. The newborn larvae were separated by filtration using a 200-mesh sieve, centrifuged at 2000 rpm for 5 minutes, and the supernatant discarded to obtain the newborn larvae. The obtained muscle larvae, adults and newborn larvae were washed 5 times with DPBS (1%, 100U / mL penicillin-streptomycin). In a 96-well cell culture plate, 100 living and morphologically intact Trichinella spiralis worms were cultured using 200μL DMEM complete medium and cultured at 37°C, 5% CO2 for 12 hours before drug addition. A blank control group and a 0.1% (v / v) DMSO negative control group were set up. Maduramicin ammonium was a 10mM DMSO stock solution, added to the worm culture medium at a final concentration of 50, 80, and 100μM, and cultured for 24, 48 and 72 hours respectively for screening. The study found that maduramicin ammonium had a good killing effect on Trichinella spiralis adults and newborn larvae, and further detailed research was carried out. The killing effect of maduramicin ammonium on adults and newborn larvae was tested when the final concentrations of 50, 80 and 100μM were cultured for 6, 12 and 24 hours respectively. Observe the compound's effect on insect morphology and structural integrity under a microscope. Insects are considered dead if they show a C-shaped or straight shape, or if they show no signs of movement after observation for more than 30 seconds. Insect mortality = average number of dead insects / average total number of insects × 100%. Each experiment is repeated three times for each drug concentration.
[0035] 1.5 Evaluation of the toxicity of maduramicin ammonium on mouse hepatic stellate cells (JS1)
[0036] JS1 mouse hepatic stellate cells were preserved by the National Hydatid Disease Laboratory. JS1 cells were seeded into 96-well cell culture plates at a density of 10,000 cells / well and incubated at 37°C for 24 hours in DMEM complete medium (10% FBS, 1% 100 U / mL penicillin-streptomycin). Maduramicin ammonium was added to the culture at final concentrations of 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 μM, and an equal volume of DMSO was added as a negative control group. After incubation for 24 hours, cell viability was detected using CCK8 cytotoxicity detection reagent. The A450 value was read on an ELISA reader, and the cell activity was calculated as follows: Cell viability = [(experimental group - blank group) / (control group - blank group)].
[0037] 1.6 Statistical analysis
[0038] SPSS1 9.0 software was used for statistical analysis, and the results were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was used to analyze the significant differences in the effects of maduramicin ammonium on the protoscolex of Echinococcus multilocularis and the muscle larvae, adults, and neonates of Trichinella spiralis. The differences were considered significant when P < 0.05.
[0039] 2. Results
[0040] 2.1 In vitro efficacy of maduramicin ammonium against Echinococcus multilocularis protoscolex
[0041] Referring to the experimental process in 1.3 above, the applicant studied the in vitro effectiveness of maduramicin ammonium against the protoscoleces of Echinococcus multilocularis, and tested the killing effects of maduramicin ammonium and ABZ on the protoscoleces of Echinococcus multilocularis at final concentrations of 5, 10, 20, 50, 80, and 100 μM at 6, 12, and 24 hours. The results showed that the killing effect of maduramicin ammonium on the protoscoleces was concentration- and time-dependent ( Figure 1 Specifically, the protoscoleces of the blank group, DMSO negative control group, and ABZ positive control group maintained 100% viability throughout the experiment ( Figure 1 A), and the morphological structure of the protoscolecus remains intact ( Figure 1 B). After 6 hours of culture, the mortality rate of the protoscoleces in the experimental group was 78.96% (P<0.01) at a concentration of 50 μM and 83.65% (P<0.01) at a concentration of 100 μM. After 24 hours of culture, the mortality rate was 100% (P<0.01). ( Figure 1 A). The morphological observation results under the optical microscope were consistent with the results of the vitality test. After 24 hours of culture, the protoscolecus of the experimental group showed extensive damage, such as collapse and shedding of the sucker, reduced calcium deposition, and obvious disappearance of the substance ( Figure 1 C) and stained blue with 0.1% methylene blue ( Figure 1 D).
[0042] 2.2 In vitro efficacy of maduramicin ammonium against Trichinella spiralis muscle larvae
[0043] Referring to the experimental process in 1.4 above, the applicant tested the killing effect of maduramicin ammonium on Trichinella spiralis muscle larvae at final concentrations of 50, 80 and 100 μM for 24, 48 and 72 h ( Figure 2 The results showed that 24 hours after adding the drug, the mortality rate of muscle larvae at a concentration of 100 μM maduramicin ammonium was 60.82% (P<0.01), and the vitality of muscle larvae decreased significantly. After 48 hours of adding the drug, the mortality rate of the larvae at a concentration of 100 μM increased to 75% (P<0.01); at 72 hours, the mortality rate of the larvae at a concentration of 100 μM reached 93.87% (P<0.01). Figure 2 A), at this time, the muscle larvae showed a clear "C"-shaped death morphology, and turned black and rigid. No signs of activity were found in the larvae after 30 seconds of observation ( Figure 2 C). The insect activity of the blank group and the DMSO group remained 100% during the entire experiment ( Figure 2 B).
[0044] 2.3 In vitro efficacy of maduramicin ammonium against adult Trichinella spiralis
[0045] Referring to the experimental process in 1.4 above, the applicant tested the killing effect of maduramicin ammonium on adult worms at final concentrations of 50, 80 and 100 μM for 6, 12 and 24 hours ( Figure 3 The results showed that 6 hours after adding the drug, the mortality rate of muscle larvae at a concentration of 100 μM maduramicin ammonium was 46.9% (P<0.05); 12 hours after adding the drug, the mortality rate of the larvae at a concentration of 100 μM increased to 80.25% (P<0.01); at 24 hours, all the larvae died at a concentration of 100 μM (P<0.01) ( Figure 3 A), the worm body is stiff and motionless and dies in a straight line ( Figure 3 C). The insect activity of the blank group and the DMSO group remained 100% during the entire experiment ( Figure 3 B).
[0046] 2.4 In vitro efficacy of maduramicin ammonium against neonatal larvae of Trichinella spiralis
[0047] Referring to the experimental process in 1.4 above, the applicant tested the killing effect of maduramicin ammonium on newborn larvae at final concentrations of 50, 80 and 100 μM for 6, 12 and 24 hours ( Figure 4The results showed that 6 hours after adding the drug, the mortality rate of newborn larvae was 66.98% (P<0.01) when the concentration of maduramicin ammonium was 100μM, and the vitality of the insects decreased significantly. 12 hours after adding the drug, all the insects died at the concentration of 100μM (P<0.01). Figure 4 A), the worm body is stiff and motionless and dies in a straight line ( Figure 4 C). The insect activity of the blank group and the DMSO group remained 100% during the entire experiment ( Figure 4 B).
[0048] 2.5 Toxic effects of maduramicin ammonium on mouse hepatic stellate cells
[0049] Referring to the experimental process in 1.5 above, the applicant added different concentrations of maduramicin ammonium to the culture medium of mouse hepatic stellate cells and cultured them for 24 hours. The results are as follows: Figure 5 As shown in the figure, the survival rate of mouse hepatic stellate cells decreased rapidly with the increase of the final concentration of maduramicin ammonium. The median lethal dose (IC50) of maduramicin ammonium on mouse hepatic stellate cells 50 ) was 2.817 μM( Figure 5 ), indicating that the dosage needs to be strictly controlled when used to treat helminthic diseases in livestock and other mammals.
[0050] As mentioned above, in the prior art, maduramicin ammonium is mainly used as an animal feed additive to prevent coccidiosis, a protozoan infection in poultry. There has been no research on its use against parasitic helminths of different species. The present invention determines the median lethal dose (IC50) of maduramicin ammonium by testing its toxic effect on mouse hepatic stellate cells in vitro. 50 ) was 2.817 μM, further confirming the high toxicity of maduramicin ammonium to mammals. With the advancement of nanotechnology and the continued in-depth screening and research of target molecules for helminths such as Echinococcus and Trichinella spiralis, further research could focus on encapsulating maduramicin ammonium in nanocapsules and assembling helminth-targeting ligand molecules on the capsule surface. These ligands would bind to the cysts through blood and lymphatic circulation, thereby reducing damage to the organism and achieving targeted insecticide.
Claims
1. Use of maduramicin ammonium in the preparation of drugs for treating helminthiasis.
2. The use of maduramicin ammonium as claimed in claim 1 in the preparation of a medicament for treating helminthiasis, characterized in that: The helminthiasis is tapeworm disease or nematode disease.
3. Use of maduramicin ammonium as claimed in claim 2 in the preparation of a medicament for treating helminthiasis, characterized in that: The tapeworm is Echinococcus multilocularis, and the nematode is Trichinella spiralis.
4. The use of maduramicin ammonium as claimed in claim 3 in the preparation of a medicament for treating helminthiasis, characterized in that: The multilocular echinococcosis is the multilocular echinococcosis protoscolecus.
5. Use of maduramicin ammonium as claimed in claim 3 in the preparation of a medicament for treating helminthiasis, characterized in that: The Trichinella spiralis is a Trichinella spiralis muscle larvae, an adult or a newborn larvae.
6. Use of maduramicin ammonium according to any one of claims 1 to 5 in the preparation of a medicament for treating helminthiasis, characterized in that: The drug for treating helminthiasis is maduramicin ammonium nanocapsule.
7. A method for killing helminthiasis in vitro, characterized in that: Maduramicin ammonium was dissolved in DMSO to prepare a DMSO stock solution, and then the DMSO stock solution was added to the culture medium of the worms.
8. The method for killing helminthiasis in vitro according to claim 7, characterized in that: The concentration of the DMSO stock solution is 10 mM.
9. The method for killing helminthiasis in vitro according to claim 8, characterized in that: The worm culture conditions were 37° C. and 5% CO 2 .
10. The method for killing helminthiasis in vitro according to any one of claims 7 to 9, characterized in that: The helminths are tapeworms or nematodes.