Application of isoquinoline alkaloids in the treatment of echinococcosis

Abstract

The present invention provides an application of isoquinoline alkaloids in treating echinococcosis. Isoquinoline alkaloids include one or more combinations of monoisoquinoline alkaloids, benzylisoquinoline alkaloids, dibenzylisoquinoline alkaloids, protoberberine alkaloids, aporphine alkaloids, propopine alkaloids, benzophenanthridine alkaloids and emetine alkaloids; and pharmaceutically acceptable salts thereof; echinococcosis is echinococcosis, including cystic echinococcosis and / or alveolar echinococcosis. The present invention uses isoquinoline alkaloids to have a good inhibitory and killing effect on Echinococcus tapeworms, especially Echinococcus larvae, and uses them to treat echinococcosis with a better therapeutic effect.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines and relates to application of isoquinoline alkaloids in treating echinococcosis. Background Art

[0002] Echinococcosis, also known as hydatidosis, is a serious zoonosis caused by the larvae of the tapeworm, Echinococcus, parasitizing humans and animals. It mainly includes cystic echinococcosis (CE) caused by Echinococcus granulosus and alveolare echinococcosis (AE) caused by Echinococcus multilocularis. In my country, echinococcosis is mainly distributed in Tibet, Xinjiang, Inner Mongolia, Qinghai, Gansu, Ningxia, Sichuan and other provinces and regions with relatively developed animal husbandry. The disease not only seriously endangers human health, but also brings heavy losses to animal husbandry.

[0003] At present, the treatment of echinococcosis mainly relies on surgery and drug chemotherapy. Since it is usually difficult to completely remove the diseased tissue by surgery, surgery combined with drug chemotherapy is generally used to reduce the risk of recurrence. Moreover, for patients who are not suitable for surgery due to contraindications, drug chemotherapy is the main treatment method. At present, the main drugs used clinically to treat echinococcosis are benzimidazole carbamate derivatives, such as albendazole and mebendazole, which work by binding to tubulin and inhibiting the formation of microtubules. Due to the low in vivo bioavailability of this type of drug, the clinical cure rate is only about 30%, and long-term or even lifelong medication is required. In view of the dilemma of lack of effective therapeutic drugs faced by echinococcosis, it is imperative to develop new anti-echinococcosis drugs. Summary of the invention

[0004] Based on the problems existing in the prior art, the first purpose of the present invention is to provide an application of isoquinoline alkaloids in the preparation of drugs for treating echinococcosis; the second purpose of the present invention is to provide a method for killing echinococcosis in vitro.

[0005] In order to solve the above technical problems, the present invention first provides an application of isoquinoline alkaloids in the preparation of a drug for treating echinococcosis.

[0006] Alkaloids are a class of alkaline nitrogen-containing organic compounds existing in nature. According to different structures, they can be divided into organic amines, pyrrolidines, pyridines, quinolines, isoquinolines, indoles, tropanes, imidazoles, quinazolones, steroids, diterpenes, purines, etc. In the screening of alkaloid drugs, the inventors found and confirmed that isoquinoline alkaloids have a good inhibitory and killing effect on Echinococcus tapeworms, especially Echinococcus larvae. Isoquinoline alkaloids are an important component of alkaloids. "New uses for old drugs" is an economical and effective classic strategy for drug development. In the prior art, isoquinoline alkaloids have many physiological activities such as antihydatid, antiarrhythmic, anticancer, analgesic and anesthesia, and their anti-Echinococcus tapeworm mechanism is still unclear.

[0007] In the above application, preferably, the isoquinoline alkaloids include one or more combinations of monoisoquinoline alkaloids, benzylisoquinoline alkaloids, bisbenzylisoquinoline alkaloids, protoberberine alkaloids, aporphine alkaloids, propionine alkaloids, benzophenanthridine alkaloids and emetine alkaloids; and pharmaceutically acceptable salts thereof (such as hydrochloride); but are not limited thereto.

[0008] In the above application, preferably, the monoisoquinoline alkaloids include scutellarine, but are not limited thereto.

[0009] In the above application, preferably, the benzylisoquinoline alkaloids include higenamine and / or magnocurine, but are not limited thereto.

[0010] The structural formula of the higenamine is as follows:

[0011]

[0012] The structural formula of the magnocurine is as follows:

[0013]

[0014] In the above application, preferably, the bis-benzylisoquinoline alkaloids include a combination of one or more of tetrandrine, fangchinoline, berbamine, cepharanthine, dauricine and liesinine; but it is not limited thereto.

[0015] The structural formula of the tetrandrine is as follows:

[0016]

[0017] The structural formula of the fangchinoline is as follows:

[0018]

[0019] The structural formula of the hydrochloride of berbamine (i.e., berbamine hydrochloride) is as follows:

[0020]

[0021] The structural formula of the cephalaenopsis is as follows:

[0022]

[0023] The structural formula of dauricine is as follows:

[0024]

[0025] The structural formula of the liesinine is as follows:

[0026]

[0027] The protoberberine alkaloids include tetrahydropalmatine and / or berberine, but are not limited thereto.

[0028] The structural formula of tetrahydropalmatine is as follows:

[0029]

[0030] The structural formula of the berberine salt is as follows:

[0031]

[0032] In the above application, preferably, the aporphine alkaloids include nuciferine and magnolamine, but are not limited thereto.

[0033] The structural formula of the nuciferine is as follows:

[0034]

[0035] The structural formula of the magnolia alkaloids is as follows:

[0036]

[0037] In the above application, preferably, the protopine alkaloid includes protopine, but is not limited thereto.

[0038] In the above application, preferably, the benzophenanthridine alkaloids include sanguinarine and / or chelerythrine; but not limited thereto.

[0039] The structural formula of the sanguinarine is as follows:

[0040]

[0041] The structural formula of the chelerythrine is as follows:

[0042]

[0043] In the above application, preferably, the emetine alkaloids include emetine and / or dehydroemetine, but are not limited thereto.

[0044] The structural formula of emetine is as follows:

[0045]

[0046] In the above application, preferably, the echinococcosis is echinococcosis, including cystic echinococcosis and / or alveolar echinococcosis; but not limited thereto.

[0047] In the above application, preferably, the echinococcosis includes Echinococcus granulosus and / or Echinococcus multilocularis; but not limited thereto.

[0048] In the above application, preferably, the Echinococcus granulosus and / or the Echinococcus multilocularis include one or more of their eggs, cysts and adults; but not limited thereto.

[0049] In the above application, preferably, the echinococcus includes one or more of its horny layer, germinal layer, germinal fossa, ascus, grandchild fossa, cyst fluid and protoscolex; but it is not limited thereto.

[0050] In the above application, preferably, the drug comprises an effective amount of isoquinoline alkaloids and pharmaceutically acceptable salts thereof (eg, hydrochloride, but not limited thereto); and optionally comprises excipients.

[0051] In the above applications, preferably, the dosage form of the drug includes tablets, granules, powders, capsules, oral solutions or injections; but not limited thereto.

[0052] On the other hand, the present invention also provides a method for killing hydatid cysts in vitro, the method comprising: dissolving isoquinoline alkaloids in dimethyl sulfoxide to prepare a dimethyl sulfoxide solution containing an effective amount of isoquinoline alkaloids; and contacting and culturing the dimethyl sulfoxide solution with hydatid cysts, thereby killing the hydatid cysts in vitro.

[0053] In the above method, preferably, the isoquinoline alkaloids include one or more combinations of monoisoquinoline alkaloids, benzylisoquinoline alkaloids, bisbenzylisoquinoline alkaloids, protoberberine alkaloids, aporphine alkaloids, propopine alkaloids, benzophenanthridine alkaloids and emetine alkaloids; and pharmaceutically acceptable salts thereof; but not limited thereto;

[0054] The hydatid cysts include Echinococcus granulosus and / or Echinococcus multilocularis; but are not limited thereto.

[0055] Among the above methods, preferably, the method is an in vitro non-therapeutic method.

[0056] In the above method, preferably, the culture condition is culturing in an incubator at 37° C. and 5% CO 2 for 1 to 6 days.

[0057] Beneficial effects of the present invention:

[0058] The present invention adopts isoquinoline alkaloids to have good inhibitory and killing effects on Echinococcus, especially Echinococcus larvae, and has a better therapeutic effect when used to treat echinococcosis. Compared with albendazole, which is clinically used in the field to treat echinococcosis, some isoquinoline alkaloids of the present invention have significant effects on Echinococcus multilocularis protoscolex in vitro, among which tetrandrine, fangchinoline, cephalothin, sanguinarine, and chelerythrine have significant effects; tetrandrine, fangchinoline, cephalothin, daurine, liesinine, sanguinarine, and chelerythrine have significant effects on Echinococcus multilocularis microcapsules in vitro. Tetrandrine has a significant effect on the inhibitory effect of Echinococcus multilocularis cysts in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The figures are comparative diagrams of the states of the protoscolex of Echinococcus multilocularis under a light microscope after the treatment of isoquinoline alkaloids, dimethyl sulfoxide (negative control group) and albendazole (positive control group) for 2 days in Example 1 of the present invention.

[0060] Figure 2 This is a morphological picture of atrophied microcysts under a light microscope after the microcysts of Echinococcus multilocularis were acted on by isoquinoline alkaloids, dimethyl sulfoxide (negative control group) and albendazole (positive control group) in Example 2 of the present invention. DETAILED DESCRIPTION

[0061] To make the purpose, technical scheme and advantages of the present invention clearer, each embodiment of the present invention will be described in detail below. However, it will be appreciated by those skilled in the art that in each embodiment of the present invention, many technical details are proposed in order to enable the reader to better understand the present invention. However, even without these technical details and various changes and modifications based on the following embodiments, the technical scheme claimed in each claim of the present invention can also be implemented. The raw materials used in the following embodiments of the present invention are conventionally commercially available unless otherwise specified, and the experimental operations used are conventional operations in the art unless otherwise specified.

[0062] Embodiment 1:

[0063] This example provides the anti-hydatid cysticercosis effect of the isoquinoline alkaloids of the present invention (higenine, magnocurine, tetrandrine, fangchinoline, berbamine hydrochloride, cepharanthin, daurine, liesinine, tetrahydropalmatine, berberine, nuciferine, magnolamine, sanguinarine, chelerythrine, emetine) and the specific experimental process is as follows:

[0064] 1. Experimental methods:

[0065] Mongolian gerbils infected with Echinococcus multilocularis were anesthetized and killed by dislocating the neck. The alveolar larvae tissue in the abdominal cavity of the gerbils was separated under sterile conditions, placed in fresh physiological saline containing 1% penicillin-streptomycin, cut into pieces and fully ground to obtain a suspension. The suspension was filtered through a 60-mesh nylon mesh, the filtrate was collected and resuspended with fresh physiological saline (containing 1% penicillin-streptomycin), the resuspended solution was filtered through a 300-mesh nylon mesh, the protoscolex on the mesh was collected, and rinsed with fresh physiological saline (containing 1% penicillin-streptomycin) 3 times. The collected protoscolex were resuspended in RPMI1640 culture medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin), an appropriate amount of the resuspended solution was taken on a slide, and the activity of the protoscolex was observed under an inverted microscope. The suspension was resuspended until its survival rate was greater than 95%, and it was diluted to about 10,000 / ml for use.

[0066] The isoquinoline alkaloids of the present invention and albendazole (positive control group) were respectively prepared into dimethyl sulfoxide (DMSO) solutions with a concentration of 50 mmol / L for later use.

[0067] 949 μl of RPMI 1640 medium (containing 10% calf serum and 1% penicillin-streptomycin), 50 μl of the stock solution of protoscolex (containing about 500 protoscolex), and 1 μl of the drug solution to be tested were added to each well of a 48-well plate, so that the final concentration was 50 μmol / L. A DMSO without drug (negative control group) was also set up. The culture plate was cultured in an incubator at 37°C and 5% CO2 for 4 days. The protoscolex status was observed under an inverted microscope every 2 days. The experiment was repeated three times.

[0068] 2. Experimental results:

[0069] The experimental results are shown in Table 1 and Figure 1 shown.

[0070] Table 1 Inhibitory effect of isoquinoline alkaloids on protoscolex of Echinococcus multilocularis

[0071] drug Protoscolex 2-day mortality (%) Protoscolex mortality rate in 4 days (%) Higenamine 0 0 Magnocurine 0 0 Tetrandrine 70～80 100 Fangchinoline 70～80 100 Berbamine Hydrochloride <5 30～40 Stephania >90 100 Dauricine 0 0 Liensinine 0 0 Tetrahydropalmatine 0 0 Berberine 0 0 Nuciferine 0 0 Magnolia alkaloids 0 0 Sanguinarine 100 - Chelerythrine 100 - emetine 0 0 Albendazole 5～10 20～30 DMSO 0 0

[0072] From Table 1 and Figure 1 It can be seen from the experimental data that some of the isoquinoline alkaloids of the present invention have an inhibitory effect on the protoscolex of Echinococcus multilocularis as compared with albendazole, which is clinically used in the field to treat echinococcosis. Among them, tetrandrine, fangchinoline, cephaladine, sanguinarine, and chelerythrine have significant effects.

[0073] Embodiment 2:

[0074] This example provides the anti-microcapsule effect of the isoquinoline alkaloids of the present invention (higenine, magnocurine, tetrandrine, fangchinoline, berbamine hydrochloride, cepharanthin, daurine, liesinine, tetrahydropalmatine, berberine, nuciferine, magnolamine, sanguinarine, chelerythrine, emetine) on Echinococcus multilocularis. The specific experimental process is as follows:

[0075] 1. Experimental methods:

[0076] Mongolian gerbils infected with Echinococcus multilocularis were anesthetized and killed by cervical dislocation. The alveolar cyst tissue in the abdominal cavity of the gerbils was separated under sterile conditions, cleaned with fresh saline containing 1% penicillin-streptomycin, cut into small pieces, and cultured in a culture dish filled with HepG2 cells (containing DMEM high-glucose culture medium, 10% fetal bovine serum, 1% penicillin-streptomycin). The cells were replaced every 2 to 3 days, and the cultured microcapsules (similar in size) were collected for later use.

[0077] The isoquinoline alkaloids of the present invention and albendazole (positive control group) were respectively prepared into dimethyl sulfoxide (DMSO) solutions with a concentration of 50 mmol / L for later use.

[0078] 999 μl of DMEM high-glucose medium (containing 10% calf serum and 1% penicillin-streptomycin), 3 microcapsules, and 1 μl of the drug solution to be tested were added to each well of a 48-well plate, so that the final concentration was 50 μmol / L. A DMSO control group without drug (negative control group) was set up at the same time. The culture plate was cultured in an incubator at 37°C and 5% CO2 for 6 days. Every 2 days, the state of the microcapsules was observed under an inverted microscope, and the number of atrophied microcapsules was recorded. The experiment was repeated twice.

[0079] 2. Experimental results:

[0080] The experimental results are shown in Table 2 and Figure 2 shown.

[0081] Table 2 Inhibitory effect of isoquinoline alkaloids on Echinococcus multilocularis microcysts

[0082]

[0083]

[0084] From Table 2 and Figure 2 It can be seen from the experimental data that some of the isoquinoline alkaloids of the present invention have an inhibitory effect on Echinococcus multilocularis microcysts that is equivalent to or even more prominent than albendazole, which is clinically used in the field to treat echinococcosis. Among them, tetrandrine, fangchinoline, cepharanthine, daurine, liesinine, sanguinarine, and chelerythrine have significant effects.

[0085] Embodiment 3:

[0086] This example provides the in vivo anti-cystic effects of two isoquinoline alkaloids (tetrandrine and berbamine hydrochloride) of the present invention on Echinococcus multilocularis. The specific experimental process is as follows:

[0087] 1. Experimental methods:

[0088] Mongolian gerbils infected with Echinococcus multilocularis were anesthetized and killed by dislocating the neck. The alveolar larvae tissue in the abdominal cavity of the gerbils was separated under sterile conditions, placed in fresh physiological saline containing 1% penicillin-streptomycin, cut into pieces and fully ground to obtain a suspension. The suspension was filtered through a 60-mesh nylon mesh, the filtrate was collected and resuspended with fresh physiological saline (containing 1% penicillin-streptomycin), the resuspended solution was filtered through a 300-mesh nylon mesh, the protoscolex on the mesh was collected, and rinsed with fresh physiological saline (containing 1% penicillin-streptomycin) 3 times. The collected protoscolex were resuspended with fresh physiological saline (containing 1% penicillin-streptomycin), an appropriate amount of the resuspended solution was taken on a glass slide, and the activity of the protoscolex was observed under an inverted microscope. The protoscolex was resuspended until its survival rate was greater than 95%, and it was diluted to about 7500 / ml for use.

[0089] 20 Kunming mice (female, 6-8 weeks old) were intraperitoneally injected with 0.2 ml of physiological saline containing protoscolex (about 1500), and after being raised in an SPF animal laboratory for 8 months, they were randomly divided into 4 groups (5 / group), of which 3 groups were drug-treated groups and 1 group was a solvent control group. The two isoquinoline alkaloids of the present invention and albendazole (positive control group) were respectively prepared into a suspension with a concentration of 10 mg / ml using a mixed solution composed of honey (Honey) and phosphate buffered saline (PBS) (ratio of 2:5), and administered by gavage at a dose of 5 ml / kg, once a day, for 40 consecutive days. After drug withdrawal, the mice were anesthetized and killed and dissected, the cysts were separated, weighed and the cyst weight was recorded, and data analysis was performed using GraphPad Prism 5.

[0090] 2. Experimental results:

[0091] The experimental results are shown in Table 3 below.

[0092] Table 3 Inhibitory effect of isoquinoline alkaloids on Echinococcus multilocularis cysts in mice

[0093] drug Capsule weight (g) Tetrandrine 6.63±1.60* Berbamine Hydrochloride 10.90±2.67 Albendazole 7.33±1.40 Honey / PBS (2:5) 13.86±2.64

[0094] *p<0.05.

[0095] It can be seen from the experimental data in Table 3 that the lower the cyst weight is than the weight of the solvent control group, the better. Compared with the solvent control group, the two isoquinoline alkaloids of the present invention both have an inhibitory effect on the multilocular Echinococcus cysts in mice. Moreover, the effect of tetrandrine of the present invention is significantly better than that of albendazole in the positive control group.

[0096] Finally, it should be noted that those skilled in the art can understand that the above-mentioned embodiments are specific examples for realizing the present invention, and in actual applications, various changes can be made in form and details without departing from the spirit and scope of the present invention.

Claims

1. Application of isoquinoline alkaloids in the preparation of drugs for treating echinococcosis; in, The isoquinoline alkaloids are bisbenzylisoquinoline alkaloids; and pharmaceutically acceptable salts thereof; The bis-benzylisoquinoline alkaloid is selected from dauricine and / or liesinine.

2. The use according to claim 1, characterized in that: The treatment of echinococcosis is manifested as an anti-microcapsule effect against Echinococcus multilocularis.

3. The use according to claim 1, characterized in that: The medicine comprises an effective amount of isoquinoline alkaloids and pharmaceutically acceptable salts thereof; and optionally comprises auxiliary materials.

4. The use according to claim 1, characterized in that: The dosage form of the drug includes tablets, granules, powders, capsules, oral solutions or injections.

5. A method for killing hydatid cysts in vitro, characterized in that: The isoquinoline alkaloids in the application according to any one of claims 1 to 4 are dissolved in dimethyl sulfoxide to prepare a dimethyl sulfoxide solution containing an effective amount of isoquinoline alkaloids; the dimethyl sulfoxide solution is contacted with hydatid cysts and cultured, thereby killing the hydatid cysts in vitro.

6. The method according to claim 5, characterized in that: The method is an in vitro non-therapeutic method.

7. The method according to claim 5, characterized in that: The culture conditions are culturing in an incubator at 37° C. and 5% CO 2 for 1 to 6 days.

8. The method according to claim 5, characterized in that: The hydatid cysts include Echinococcus granulosus and / or Echinococcus multilocularis.

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