CN109420172B - Application of a kind of inhibitor in preparing medicine for treating cystic hydatid disease - Google Patents
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Abstract
The invention relates to application of an inhibitor in preparation of a medicine for treating cystic echinococcosis. Specifically, the invention discloses the application of an inhibitor for preparing a medicament for treating cystic echinococcosis; and the inhibitor is an inhibitor selected from the group consisting of: a deubiquitinase inhibitor, an EGFR inhibitor, or a combination thereof. The inhibitor can effectively kill the echinococcus granulosus (especially echinococcus granulosus metacercaria), and further realize the treatment of cystic echinococcosis.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of an inhibitor in preparation of a medicine for treating cystic echinococcosis.
Background
Echinococcus granulosus (e.g. grandiulosus, E.g) and echinococcus multocida (e.g. multilaocuraris, E.m) both parasitize causing cystic Echinococcosis (cysis Echinococcosis, CE) and Alveolar Echinococcosis (AE). The high incidence areas of echinococcosis in China are mainly prevalent in alpine meadow areas, pasturing areas with cold climate, drought and little rain, and semi-farming and semi-pasturing areas, so that areas such as Sinkiang, Tibet, Gansu, Ningxia are serious, and Sinkiang is one of the high incidence areas of echinococcosis.
Dogs and foxes usually eat mice which are easy to eat vegetation polluted by animal excrement, insect eggs, and the like, so that the life history of the dogs and the foxes is completed. After the fox and other animals swallow the viscera of the mice or animals with the cyst larvae, the protocephalic larvae grow into adults after about 45 days, and the pregnancies and the ova are discharged with the feces, so that the human is infected by eating the ova by mistake. With the development of the society at present, the population mobility is increased, the number of pet dogs and foxes is increased, and the echinococcosis area is gradually spread, which will seriously affect the human health. Most (> 90%) of echinococcosis is cystic echinococcosis caused by echinococcus granulosus larvae infection, and there are four methods mentioned for the treatment and management of cystic echinococcosis according to WHO-IWGE: surgery, drug chemotherapy, minimally invasive and observation.
At present, the treatment of echinococcosis is still preferred from surgical operation, and the clinical effect that the medical treatment can improve the surgical cure rate and reduce the recurrence rate is continuously determined. The first choice of chemotherapeutic drugs for treating echinococcosis is benzimidazole drugs, and the representative drugs are: the two drugs are only approved benzimidazole drugs, the drugs aim at the beta-tubulin of the parasite which is highly similar to the beta-tubulin of human beings, the taking of the albendazole has slight influence on the secretory excretion function and the synthesis function of the liver and causes slight damage to the liver in some studies, and the clinical curative effects of 212 cases and 116 cases of the albendazole emulsion show that the clinical curative effect exceeds the best level of the current echinococcosis drugs, the curative effect is reliable and stable, but various adverse reactions cause poor clinical curative effects.
Therefore, the development of new target chemical drugs is not slow. Therefore, the method has great significance for actively searching for new drugs and action targets thereof and non-operative treatment methods for preventing and treating the echinococcosis. In recent years, research and development of drugs for treating hydatid have been conducted from different angles, and research has been conducted mainly from the aspects of finding new drugs and the like.
Studies have now demonstrated that many deubiquiti2 natingenzymes (DUBs) are widely present in cells, and are mainly classified into 5 types, mainly the ubiquitin carboxyl-terminal hydrolase family and the ubiquitin-specific processing enzyme family. These different types of deubiquitinases can hydrolyze the link between ubiquitin chains on the substrate protein, play a role in deubiquitinating, and carry out reverse regulation on protein degradation, thereby affecting the function of the protein. Deubiquitinating enzymes can affect or regulate many cellular physiological and pathological processes such as cell growth and development, signal transduction, and neuropathy or tumors. However, a DUB inhibitor (e.g., Degrasyn) with antitumor effect has not been reported to be applicable to the treatment of cystic echinococcosis.
EGFR (epidermal Growth Factor receptor) is a receptor for cell proliferation and signaling by the Epidermal Growth Factor (EGF). EGFR belongs to the ErbB receptor family, which includes EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3(ErbB-3), and Her 4 (ErbB-4). EGFR is also known as HER1, ErbB1, and mutation or overexpression typically causes tumors. EGFR is a glycoprotein belonging to tyrosine kinase type receptor, and has a penetrating cell membrane and a molecular weight of 170 KDa. EGFR is located on the surface of cell membranes and is activated by binding to ligands, including EGF and TGF α (transforming growth factor α). Research shows that high expression or abnormal expression of EGFR exists in a plurality of solid tumors, so that the effect of treating tumors is achieved, but research on the EGFR in echinococcosis is not carried out.
Disclosure of Invention
The invention aims to provide application of an inhibitor (such as a de-ubiquitinase inhibitor Degrasyn and/or an EGFR inhibitor tyrphosin 9) in preparation of a medicament for treating cystic echinococcosis.
In a first aspect of the invention, there is provided the use of an inhibitor for the manufacture of a medicament for the treatment of cystic echinococcosis;
and the inhibitor is an inhibitor selected from the group consisting of: a deubiquitinase inhibitor, an EGFR inhibitor, or a combination thereof.
In another preferred embodiment, the deubiquitinase inhibitor is a compound shown as a formula A, or an isomer, a solvate or a pharmaceutically acceptable salt thereof,
wherein Br on the pyridine ring is optionally substituted with a halogen selected from the group consisting of: F. cl, I, H on the pyridine ring is optionally substituted with a halogen selected from the group consisting of: F. cl, Br, I, the number of halogen substituents on the pyridine ring is 1-4 (e.g. 1, 2, 3, 4).
In another preferred embodiment, the deubiquitinase inhibitor is
In another preferred embodiment, the EGFR inhibitor is a compound selected from the group consisting of an ester thereof, an isomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof,
in another preferred embodiment, the cystic echinococcosis is a disease caused by echinococcus granulosus infection.
In another preferred embodiment, the echinococcus granulosus is selected from the group consisting of: echinococcus granulosus larvae, Echinococcus granulosus embryonic cells, Echinococcus granulosus germinal layer, or a combination thereof.
In another preferred embodiment, the echinococcus granulosus larvae are echinococcus granulosus metacercaria.
In a second aspect of the invention, there is provided a formulation for use in the treatment of cystic echinococcosis, the formulation comprising:
1) a compound represented by formula A, or an isomer thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof as a first pharmaceutically active ingredient,
wherein Br on the pyridine ring is optionally substituted with a halogen selected from the group consisting of: F. cl, I, H on the pyridine ring is optionally substituted with a halogen selected from the group consisting of: F. cl, Br, I, the number of halogen substituents on the pyridine ring is 1-4 (such as 1, 2, 3, 4); and/or
2) A compound selected from the group consisting of an ester thereof, an isomer thereof, a solvate thereof, and a pharmaceutically acceptable salt thereof as a second pharmaceutically active ingredient,
3) optionally a third pharmaceutically active ingredient, different from the first and second pharmaceutically active ingredients, for use in the treatment of cystic echinococcosis; and
4) a pharmaceutically acceptable carrier.
In another preferred embodiment, the third pharmaceutically active ingredient is selected from the group consisting of: a monoclonal antibody, a liposome, a microemulsion, or a combination thereof.
In another preferred embodiment, the formulation is in a dosage form selected from the group consisting of: tablet, granule, capsule, oral liquid, and injection.
In a third aspect of the invention, a method for killing echinococcus granulosus in vitro is provided, wherein the first component and/or the second component is contacted with the echinococcus granulosus, thereby killing the echinococcus granulosus;
wherein the first component is a compound shown as a formula A, or an isomer, a solvate or a pharmaceutically acceptable salt thereof,
wherein Br on the pyridine ring is optionally substituted with a halogen selected from the group consisting of: F. cl, I, H on the pyridine ring is optionally substituted with a halogen selected from the group consisting of: F. cl, Br, I, the number of halogen substituents on the pyridine ring is 1-4 (such as 1, 2, 3, 4);
the second component is a compound selected from the group consisting of an ester thereof, an isomer thereof, a solvate thereof, and a pharmaceutically acceptable salt thereof,
in another preferred example, the echinococcus granulosus is selected from the group consisting of: echinococcus granulosus larvae, Echinococcus granulosus embryonic cells, Echinococcus granulosus germinal layer, or a combination thereof.
In another preferred embodiment, the echinococcus granulosus larvae are echinococcus granulosus metacercaria.
In another preferred embodiment, the method is an in vitro non-therapeutic method.
In a fourth aspect of the present invention, there is provided a method of treating cystic echinococcosis, the method comprising the steps of: administering a therapeutically effective amount of the first component and/or the second component to a patient in need thereof; wherein the first component is a compound shown as a formula A, or an isomer, a solvate or a pharmaceutically acceptable salt thereof,
wherein Br on the pyridine ring is optionally substituted with a halogen selected from the group consisting of: F. cl, I, H on the pyridine ring is optionally substituted with a halogen selected from the group consisting of: F. cl, Br, I, the number of halogen substituents on the pyridine ring is 1-4 (such as 1, 2, 3, 4);
the second component is a compound selected from the group consisting of an ester thereof, an isomer thereof, a solvate thereof, and a pharmaceutically acceptable salt thereof,
in another preferred embodiment, the "subject in need thereof" includes both human and non-human mammals (e.g., mice, rats).
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Detailed Description
After long-term and intensive research, the inventors unexpectedly found that deubiquitinase inhibitors (such as Degrasyn) and/or EGFR inhibitors (such as Tyrphostin9) have excellent activity of killing echinococcus granulosus, so that the deubiquitinase inhibitors and/or EGFR inhibitors can be used for preparing medicines for treating cystic echinococcosis, and provide a new choice for treating the cystic echinococcosis. On this basis, the inventors have completed the present invention.
Deubiquitinase inhibitors
Deubiquitinating refers to a negative regulation process of protein ubiquitination mediated by deubiquitinase, and plays an extremely important role in the maintenance of homeostasis in cells, the stabilization or degradation of proteins, and signal transduction pathways in cells. In human tumors, deubiquitinase inhibitors inhibit the growth of tumors by interfering the activity thereof, and become a hotspot for developing novel antitumor drugs.
The invention provides an application of a deubiquitinase inhibitor in preparing a medicine for treating cystic echinococcosis, wherein the deubiquitinase inhibitor is a compound shown as a formula A, or an isomer, a solvate or a pharmaceutically acceptable salt thereof,
wherein Br on the pyridine ring is optionally substituted with a halogen selected from the group consisting of: F. cl, I, H on the pyridine ring is optionally substituted with a halogen selected from the group consisting of: F. cl, Br, I, the number of halogen substituents on the pyridine ring is 1-4 (e.g. 1, 2, 3, 4).
A typical deubiquitinase inhibitor is Degrasyn, which has the structural formula
Degrasyn is a selective inhibitor, also has inhibitory effect on Bcr/Abl, and is also a JAK2 sensor (without affecting 20S proteasome) and activator of transcription (STAT). It was found that degranyn was more effective in reducing leukemic cell colony formation and was effective against primary leukemic cells with the T315I mutant. In vitro studies showed that the compound has a potent inhibitory effect on A375 melanoma established in nude mice.
EGFR inhibitors
The invention provides an application of an EGFR inhibitor in preparing a medicament for treating cystic echinococcosis, wherein the EGFR inhibitor is a compound selected from the following group, or an ester, an isomer, a solvate or a pharmaceutically acceptable salt thereof,
in the invention, five compounds, namely Afatinib (BIB2992), AG-490 (tyrphosin B42), Pelitinib (EKB-569), AG-1478 (tyrphosin AG-1478) and CNX-2006 are EGFR inhibitors, and the death rate of the protocercaria reaches over 95% after 24 hours of a primary killing experiment on the protocercaria.
A typical EGFR inhibitor is Tyrphostin9, which has a structural formula
Tyrphostin9 was the first EGFR inhibitor, with an IC50 of 460 μ M, acting more effectively on PDGFR and an IC50 of 0.5 μ M. Tyrphostin9 inhibits herpes simplex virus type 1 (HSV-1) replication, and Tyrphostin9 (HSV-1)<800nm) induced a reduction in protein synthesis, not dose dependent. Tyrphostin 9(1 μ M) increased the outer MAPT exon 10.
Cystic echinococcosis
Echinococcosis, also known as echinococcosis, is a parasitic disease of zoonosis, in which cystic echinococcosis is distributed worldwide and infects echinococcosis by spreading intermediate hosts including humans, ungulates, mice, etc. by eating eggs by mistake. Many organs of the whole body can occur, and liver and lung are common. The early stage of the infection does not have any clinical symptoms, the clinical symptoms of the echinococcosis are generally no obvious symptoms in the early stage of the infection, the cyst grows and presses lung tissues and bronchus, and patients can have chest pain, cough, blood phlegm, shortness of breath and even dyspnea. The clinical manifestations are paroxysmal cough and dyspnea. Allergic reactions, even shock, can accompany them. If the large blood vessel is ruptured, hemoptysis may occur. The incubation period of cystic echinococcosis is about 1-30 years, and the disease course varies according to the organ occupied by echinococcus. It is generally considered that the treatment is mainly performed by surgery, and the importance of drugs as important auxiliary means is increasing.
Compared with the prior art, the invention has the following main advantages:
(1) the invention provides application of a deubiquitinase inhibitor (such as Degrasyn) and/or an EGFR inhibitor (such as tyrphosin 9) in preparation of a medicine for treating cystic echinococcosis, and provides a new choice for treating cystic echinococcosis;
(2) formulations containing the deubiquitinase inhibitor (e.g., degranyn) and/or the EGFR inhibitor (e.g., Tyrphostin9) are effective in killing protozoa larvae of parasites that cause cystic echinococcosis (e.g., echinococcus granulosus).
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
EXAMPLE 1 pharmacodynamic experiment of drug screening
1. Content of the experiment
1.1 Echinococcus granulosus Procoenurosis in vitro killing
Collecting the metacercaria: sheep livers from sheep with hydatid infusorian were purchased from the Wuluqi slaughterhouse and brought back to the laboratory. The serum attached to the surface of the sheep liver is washed clean by water, and then the adipose tissues on the surface of the sheep liver are removed as much as possible by scissors. The surface of the goat liver was wiped with 75% alcohol, the cyst fluid in the cyst was aspirated with a 50ml syringe, placed in a 50ml centrifuge tube, and a visual window of about 2cm was cut along the junction of the cyst and liver tissues with ophthalmic scissors. The inner sac of the cyst was carefully peeled off with forceps and placed in a dish containing physiological saline. Collecting the metacercaria naturally settled from the cyst fluid and washed on the inner cyst wall, removing the host tissue in the sediment by using gauze, digesting the metacercaria in a constant-temperature water bath kettle at 37 ℃ for 30min according to the volume of 1:10 and 1 percent of pepsin (pH 2.0), observing the metacercaria to be in a fine sand shape by naked eyes, and pouring the metacercaria under a microscope to observe no small microcapsule, namely the metacercaria is completely digested.
The digested protozoa are washed with normal saline, and the protozoa are generally washed 10 times with 10 times the volume of normal saline. Prepare 1640 medium containing 20% fetal bovine serum (all reagents used were purchased from Hyclon, such as fetal bovine serum, 1640 medium, and penicillin), and dilute the metacercaria with the prepared medium to a final concentration of 250/ml. The mixed metacercaria is added into a 96-well round-bottom plate by using a 200ul arraying gun, and whether the quantity of metacercaria in each well is uniform or not is observed under a mirror, if more or less metacercaria exist, the adjustment is needed.
The ABZ group served as a positive control drug (albendazole available from sigma, usa) with 3 replicate wells set. DMSO was used as a negative control drug, and 3 replicate wells were also set. 378 different small-molecule drugs are added into corresponding holes, and the drugs are mixed uniformly immediately after the drugs are added, so that the phenomenon that the original metacercaria is immediately killed due to the fact that the drugs wrap the original metacercaria is avoided.
Survival was observed for 2 hours, 4 hours, 6 hours, 12 hours, 24 hours and recorded by photographing. After 24 hours, staining with 0.01% methylene blue dye for 1min, washing metacercaria until the background is transparent, and taking a picture to record.
And after the observation is finished, counting the death number of the metacercaria, and calculating the death rate of the metacercaria. The drug with the mortality rate higher than that of the positive control group is obtained, and the steps are repeated once by using the drug with low concentration.
1.2 medicine LD50(i.e., median lethal dose) calculation
The screened drugs were serially diluted 10-fold with DMSO in a 96-well plate, and the drugs from high to low concentration were added to the corresponding wells, and 100. mu.l of the medium was added to each well using an 8-well row gun, so that the drugs were well mixed in the medium. The metacercaria is diluted to 500 per ml by using the culture medium, and 100 mul of the culture medium is added into each hole by using a discharging gun after shaking up, so that the number of the metacercaria in each hole is about 50. 5% CO at 37 ℃2Culturing in a constant temperature incubator for 24 hours, staining with methylene blue, and recording and observing the survival condition of the metacercaria.
The mortality rate of the metacercaria at each concentration is counted, and the result is calculated according to the Reed-Muench two-handed method.
After 24 hours, staining is carried out for 1min, and statistics of photographing is observed, so that the mortality rate of the metacercaria in the Degrasyn administration group almost reaches 100% when the concentration is 10 mu M, and the mortality rate is far higher than that of the albendazole group. When the concentration is adjusted to 5 mu M, the death rate of the Degrasyn group is about 95 percent, and the killing effect is still better than that of the albendazole group. At concentrations as low as 1 μ M, the albendazole group still did not kill as well as the Degrasyn group.
Then determining the LD50 of Degrasyn killing metacercaria, and obtaining the LD of Degrasyn on metacercaria praecox after staining observation50The in vitro effect of Degrasyn on Echinococcus granulosus metacercaria obtained by the experiment is better than that of albendazole, and the in vitro effect is 2.5 mu M.
In addition, the observation and statistics also show that the drug concentration is 10 mu M, and the mortality rate of the tyrphosin 9 group is far greater than that of the albendazole group. At a concentration of 5. mu.M, the mortality rate of the tyrphosin 9 group metacercaria is about 95 percent and is higher than that of albendazole. Finally, the concentration is adjusted to 1 mu M, and the mortality rate of the Tyrphostin 91 mu M group is still higher than that of the albendazole group metacercaria.
Tyrphostin kills metacercaria LD50The LD of tyrphosin 9 drug against Echinococcus granulosus metacercaria was calculated50As low as 2. mu.M, the tyrphosin 9 has a certain killing effect on Echinococcus granulosus metacercaria, and the tyrphosin 9 has a better killing effect in vitro than albendazole.
Example 2 in vitro killing of Encapsulated capsules (Degrasyn)
Directly injecting the metacercaria into abdominal cavity, hepatic portal vein or liver parenchyma of the murine intermediate animal, or injecting cysts transformed by in vitro cultured metacercaria into the murine intermediate animal. The mice were dissected and the cysts removed. Sterile and intact capsules were dispensed into 6-well plates and different concentrations of degranyn were added. Dynamically observing whether the cyst is cracked, collapsed, and the germinal layer is shed within one week, and determining the in vitro curative effect of the medicine.
Capsules with the diameter range of 5 mm-9 mm are taken, the concentration obtained by experiments is 20ug/ml, the albendazole group capsule death rate is 0%, and the death rate of Degrasyn is 60%. The in vitro experiment of the cyst shows that the in vitro killing cyst of Degrasyn has better effect than albendazole.
EXAMPLE 3 Encapsulated in vitro killing (Tyrphostin9)
Directly injecting the metacercaria into abdominal cavity, hepatic portal vein or liver parenchyma of the murine intermediate animal, or injecting cysts transformed by in vitro cultured metacercaria into the murine intermediate animal. The mice were dissected and the cysts removed. Sterile and intact capsules (ranging from 5mm to 10mm in diameter) were dispensed into 6-well plates and Tyrphostin9 was added at various concentrations. Dynamically observing whether the cyst is cracked, collapsed, and the germinal layer is shed within one week, and determining the in vitro curative effect of the medicine.
Experimental observations showed that at a drug concentration of 20ug/ml, the Capsule mortality rate was 60% for the Tyrphostin9 group, and only 0% for the albendazole group. The result shows that Tyrphostin9 has better killing effect than albendazole in vitro.
Example 4 in vivo experiments in mice (Degrasyn and/or Tyrphostin9)
Directly injecting the metacercaria into abdominal cavity, hepatic portal vein or liver parenchyma of the murine intermediate animal, or injecting cysts transformed by in vitro cultured metacercaria into the murine intermediate animal. After 1-10 months after inoculation, different concentrations of drugs (Degrasyn and/or Tyrphostin9) were administered orally or by injection to these experimental animals, and 1-50 weeks after administration to the animals, the experimental animals were dissected and the efficacy of the drugs was determined by the decrease in the number of cysts, the change in the size or structure of the cysts.
In view of the above, the present invention discloses for the first time the study of DUB inhibitors (e.g. degranyn) and/or EGFR inhibitors (e.g. tyrphosin 9) as drugs for the treatment of cystic echinococcosis.
In vivo and in vitro experimental data show that Degrasyn has an in vitro killing effect on metacercaria, the killing effect of Degrasyn on metacercaria is superior to that of albendazole on metacercaria, and the Degrasyn also has an obvious killing effect on cysts, so that the DUB inhibitor Degrasyn has a treatment effect on cystic echinococcosis, namely the DUB inhibitor can be used as a novel echinococcosis-resistant medicament for cystic echinococcosis, and a new way is provided for treating the cystic echinococcosis.
Furthermore, the above results also show that: degrasyn has obvious killing effect on Echinococcus granulosus metacercaria in low concentration and short time, and provides a new way for treating cystic echinococcosis.
In addition, the data of in vitro experiments show that: the tyrphosin 9 has an in-vitro killing effect on protocysticercosis, which indicates that the EGFR inhibitor has a treatment effect on cystic echinococcosis, i.e. the EGFR inhibitor can be used as a novel echinococcosis resisting drug for cystic echinococcosis, thereby providing a new way for treating the cystic echinococcosis.
Furthermore, the above results also show that: tyrphostin9 has significant killing effect on Echinococcus granulosus metacercaria in low concentration and short time, and provides a new approach for treating cysticercosis.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
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| CN112933090B (en) * | 2021-03-16 | 2022-11-08 | 军事科学院军事医学研究院军事兽医研究所 | Application of deubiquitinase inhibitor in preparation of anti-rabies virus medicine |
| CN115737817A (en) * | 2022-11-14 | 2023-03-07 | 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) | Application of tyrosine kinase inhibitor in preparation of echinococcosis treatment drug |
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