Application of CA-170 in preparation of medicine for treating echinococcosis multilocularis
By using an oral small molecule CA-170 formulation targeting VISTA and PD-L1, the challenges of immunosuppression and liver parasitic lesions in echinococcosis have been addressed, achieving effective treatment of echinococcosis and reducing lesions and liver damage.
Patent Information
- Application Number
- CN202511066949.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-31
- Publication Date
- 2025-11-18
AI Technical Summary
Current technologies are insufficient to effectively treat echinococcosis, especially since immunosuppression and liver parasitic lesions caused by echinococcosis infection are difficult to control. Conventional drug treatments have limited effectiveness, and surgical resection is difficult and ineffective.
The oral small molecule dual inhibitor CA-170 targets the immune checkpoints VISTA and PD-L1 to enhance T cell function and is formulated into an oral dosage form for the treatment of multilocular echinococcosis.
It significantly reduces the number and weight of lesions in multilocular echinococcosis, lowers the proportion of MDSCs, and alleviates liver damage, providing an efficient treatment option and laying the foundation for clinical treatment.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biological medicine, in particular to the application of VISTA and PD-L1 double inhibitor CA-170 in the preparation of a drug for treating alveolar echinococcosis. BACKGROUND
[0002] Alveolar echinococcosis (AE), also known as cystic echinococcosis, is a serious zoonosis caused by the larva of Echinococcus multilocularis. AE is also known as "worm cancer" due to its tumor-like growth and metastatic ability. Clinical data shows that the mortality rate of AE patients without treatment or insufficient treatment is about 90% within 10-15 years. The conventional treatment strategy for this disease is mainly surgical resection, supplemented by drug treatment. However, most patients have multiple lesions and often diffuse distribution in the liver, so only 30% of patients can be surgically resected, and the effect of surgical treatment is often poor. In addition, the current albenzda drug treatment has very limited effect, making the disease difficult to treat. Therefore, the development of new and efficient therapeutic drugs is very urgent.
[0003] Echinococcosis infection can cause strong immune suppression in the host, which is a key problem in the prevention and treatment of the disease. Studies have found that echinococcosis infection can inhibit the host's innate immune response by promoting the polarization of macrophage M2, inducing the differentiation of suppressive dendritic cells (DC), and depleting natural killer T cell (NKT) function, etc., breaking through the host's immune barrier, and then forming a chronic and persistent infection. In the current research on echinococcosis, the blockade therapy targeting immune checkpoints such as PD-1 has achieved certain effect in clinical practice (can reduce T lymphocyte apoptosis and exhaustion, and enhance the immune function of the body), but has no obvious inhibitory effect on the growth of liver parasitic lesions, which suggests that there are other key pathways in echinococcosis infection.
[0004] Studies have found that a large number of myeloid-derived suppressor cells (MDSCs) are recruited at the site of liver parasitic lesions after Echinococcus multilocularis infection through single-cell sequencing data and flow cytometry analysis. The key molecules and pathways of MDSC-mediated immune suppression in the current echinococcosis are not clear, which greatly limits the development and clinical application of MDSCs as a potential therapeutic target. Further analysis found that MDSCs specifically highly express the immune checkpoint gene VISTA (V-set immunoglobulin domain-containing suppressor of T cell activation).
[0005] CA-170 is an oral small molecule inhibitor that targets the immune checkpoints VISTA and the programmed death ligands PD-L1 and PD-L2, thereby enhancing the proliferative capacity of T cells and the production of activating cytokines. In existing preclinical studies, CA-170 has shown good anti-tumor activity in multiple mouse tumor models. However, there is no report on the research of CA-170 as a treatment drug for echinococcosis. SUMMARY
[0006] Based on the above, the purpose of the present application is to provide a new use of the oral small molecule dual antagonist CA-170 selectively targeting PD-L1 and VISTA, i.e. the use of CA-170 in the preparation of a drug for treating multilocular echinococcosis.
[0007] The molecular formula of CA170 used in the present application is C 12 H 20 N6O7, CAS No. 1673534-76-3, and the structural formula is as shown below:
[0008]
[0009] The present application provides the use of CA-170 in the preparation of a drug for treating multilocular echinococcosis, which is the cyst development of protocephalum caused by multilocular echinococcus infection (protocephalum continuously produces new vesicles through exogenous budding reproduction in the host body, and these vesicles are connected and aggregated, often forming a structure similar to a tumor), or liver damage caused by multilocular echinococcus infection.
[0010] The dosage form of the drug is an oral dosage form. When CA-170 is administered, it is first dissolved in DMSO and then diluted with physiological saline before administration. The dose of CA-170 in the drug is not less than 10 mg / kg.
[0011] The present application also provides a pharmaceutical composition for treating multilocular echinococcosis, comprising CA-170 or a pharmaceutically acceptable salt or hydrate of the salt thereof, and a pharmaceutically acceptable excipient.
[0012] The dosage form of the pharmaceutical composition is an oral dosage form, such as tablets, granules, powders, capsules, oral liquids or oral preparations.
[0013] The present application has the following advantages:
[0014] The application provides application of CA170 in preparation of drugs for treating multilocular hydatidosis, so as to realize effective treatment of Echinococcus multilocularis infection. The application takes C57 / 6J mice infected with multilocular hydatid through hepatic portal vein for 40 days as research objects, and orally administers CA-170 small molecules, the concentration of the CA-170 is not less than 5.6 μM, once every three days, treatment for one month, and the drug efficacy of CA-170 on cyst development and lesion growth of protoscolex in the Echinococcus multilocularis infected mice is detected. The results show that, compared with the solvent control group, the lesions in the CA-170 treatment group mice are reduced, the number and weight of the hydatid cysts are significantly reduced, which indicates that CA170 has good anti-Echinococcus granulosus effect, and can reduce the proportion of MDSC caused by infection and alleviate the liver damage caused by infection. The application scheme provided by the application will provide technical support for efficient prevention and treatment of multilocular hydatidosis, and lay a foundation for clinical treatment of CA-170 on multilocular hydatidosis. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1 The weight change results of the mice in each group after CA-170 treatment;
[0016] Figure 2 The statistical results of the cyst inhibition rate of each experimental group after CA-170 treatment;
[0017] Figure 3 The liver pictures of the mice in each group after CA-170 treatment;
[0018] Figure 4 The statistical results of the liver cyst weight of the mice in each group after CA-170 treatment. DETAILED DESCRIPTION
[0019] The application will be described in detail below with reference to the accompanying drawings and specific examples.
[0020] Example 1
[0021] 1. The drug efficacy of CA-170 on cyst development and lesion growth of protoscolex in the Echinococcus multilocularis infected mice
[0022] 1.1 The establishment of a mouse model of liver multilocular hydatidosis by injecting protoscolex through hepatic portal vein
[0023] The infected gerbils were sacrificed by cervical dislocation and the scoleces were isolated under sterile conditions after the gerbils were soaked with 75% alcohol. The cysts were peeled from the gerbil tissues, and the residual tissues were removed. The cysts were repeatedly washed in pre-cooled PBS (containing 100 U / mL penicillin and 100 U / mL streptomycin) buffer for 3 times. The cysts were cut into small pieces with ophthalmic scissors, and the mixture was passed through a 80-mesh copper screen. The worms were allowed to settle naturally, and the supernatant was discarded until the solution was clear. The cysts were repeatedly washed for 3 times, and diluted to a concentration of 5000 / mL.
[0024] Eighteen 8-week-old C57BL / 6 mice were randomly divided into two groups, 6 mice in the negative control group and 12 mice in the infection group. The mice in the infection group were injected with 100 μL of the suspension (containing 5000 scoleces) through the portal vein, and the mice in the control group were injected with the same volume of PBS through the portal vein.
[0025] 1.2 CA-170 treatment
[0026] 1.2.1 Experimental grouping: 12 mice infected with E. granulosus for 40 days were randomly divided into two groups, 6 mice in each group, namely the solvent control group and the CA-170 treatment group.
[0027] 1.2.2 CA-170 preparation: CA-170 was first dissolved in DMSO at a concentration of 28.5 mg / mL, and then dissolved in normal saline to a working concentration of 1 mg / mL.
[0028] 1.2.3 Route and time of administration: the mice were orally administered with CA-170 by gavage, and the drug dose was set as 10 mg / kg. At the same time, the mice in the solvent control group were administered with the same dose of DMSO in normal saline by gavage. The administration was performed every 3 days, and the administration period lasted for 30 days.
[0029] 1.2.4 Data statistical method: after the treatment, the weight of the liver and the weight of the cysts after the liver was removed were accurately counted.
[0030] The results showed that compared with the solvent control group, the liver morphology of the CA-170 treatment group was basically complete ( Figure 1 ), and the number and size of the cysts parasitized in the liver were significantly reduced, indicating that CA-170 could significantly inhibit the growth of the cysts ( Figure 2 ).
[0031] 2. CA-170 treatment reduces the proportion of MDSC caused by E. granulosus infection
[0032] 2.1 Preparation of liver non-parenchymal cell suspension
[0033] The whole liver was removed from the dissected mouse, and after removing the cyst, the liver was placed in a 5 mL centrifuge tube. The liver was cut into pieces using dissecting scissors, and 4 mL of 0.2% collagenase II digestion solution was added. The mixture was incubated at 37°C for 30 min, and the mixture was inverted and mixed every 5 min during the incubation. After the digestion was completed, the mixture was passed through a 70 μm cell strainer to obtain a single cell suspension. The single cell suspension was centrifuged at 50 x g for 10 min, and the supernatant was removed. The cell pellet was centrifuged at 400 x g for 10 min, and 1 mL of red blood cell lysis solution was added to the cell pellet. The mixture was gently mixed and incubated at room temperature for 5 min. After the lysis was completed, 9 mL of PBS was added to terminate the lysis, and the mixture was centrifuged at 400 x g for 10 min. The supernatant was removed, and the cell pellet was resuspended in an appropriate amount of PBS. The cell concentration was adjusted to 1 x 10 6 / mL using a cell counting plate.
[0034] 2.2 Flow cytometry cell surface antibody staining
[0035] 100 μL of the cell suspension (1 x 10 5 ) was added to each flow tube. First, 0.2 μL of FVD was added to each tube, and the mixture was incubated at room temperature in the dark for 10 min. The cells were washed with PBS, and the supernatant was removed after centrifugation. 100 μL of CD16 / 32 blocking solution (1 μg / 10 6 cells) was added, and the mixture was incubated at room temperature in the dark for 30 min. After the blocking was completed, the following fluorescent antibodies were sequentially added for cell surface marker staining: CD45-FITC, CD11b-PC5.5, and Gr-1-PE. The mixture was incubated at room temperature in the dark for 60 min, and the cell suspension was gently mixed during the incubation to ensure that the antibodies were fully combined with the cells. After the staining was completed, the cells were washed with PBS for 2-3 times, and the supernatant was removed after each centrifugation. Finally, the cells were resuspended in 400 μL of PBS. The flow cytometer was used for detection, and the appropriate detection channel was selected according to the fluorescently labeled antibodies. The expression of the cell surface markers was analyzed.
[0036] 2.2 Flow cytometry experiment procedure and analysis In the flow cytometry gating principle, P1 gate was used to circle the main cell population to exclude cell debris; P2 gate was used to exclude cell adhesion; P3 gate was used to circle the live cells; P4 gate was used to circle the CD45 positive cells; P5 gate was used to circle the CD11b positive cells; and P6 gate was used to circle the Gr1 positive cells.
[0038] 2.3 Experimental results
[0039] Compared with the healthy control group, the number of MDSCs (CD11b+Gr1 + ) at the liver lesion site significantly increased after Echinococcus multilocularis infection. After treatment with CA-170, the number of MDSCs at the liver lesion site was reduced compared with the solvent control group.Figure 3 ).
[0040] 3. CA-170 treatment reduces liver damage caused by Echinococcus multilocularis or cysticercus infection
[0041] 3.1 Serum preparation
[0042] The mouse peripheral blood was collected in a 1.5 mL centrifuge tube, 37°C for 60 min, 4°C overnight, 3000 rpm centrifugation for 10 min, and the separated serum was transferred to a clean centrifuge tube.
[0043] 3.2 Alanine aminotransferase (ALT) content determination
[0044] The ALT detection kit was purchased from Shanghai Yuan Ye Biological Technology Co., Ltd. (S30HR196604).
[0045] Experimental procedure:
[0046] Preparation of ALT standard curve: Take one branch of pyruvic acid standard, accurately add 1 ml of pyruvic acid standard diluent, mix well, and prepare pyruvic acid standard (100 mmol / L), store at 4°C for standby. Before use, take an appropriate amount of pyruvic acid standard (100 mmol / L), mix according to the ratio of pyruvic acid standard (100 mmol / L): pyruvic acid standard diluent = 1:49, which is pyruvic acid standard working solution-pyruvic acid standard (2 mmol / L), and prepare the standard curve according to the ALT karmen enzyme activity units 0, 24, 61, 114, 190 (KU). Set parallel detection holes and calculate the average value. Mix well, add 20 μL of dinitrophenylhydrazine color developing solution to each tube, incubate at 37°C for 20 min, then add 200 μL of ALT color developing base solution (1x), mix well. Place at room temperature for 5 min, adjust to zero with distilled water, and measure the absorbance of each hole at 505 nm with an enzyme marker. Subtract the absorbance of the "0" hole from the absorbance of each hole, and plot the difference in absorbance (vertical coordinate) and the corresponding karmen enzyme activity units (horizontal coordinate).
[0047] ALT enzymatic reaction: set control wells, test wells, and detection wells. Add 4 μL of the serum to be tested and 20 μL of ALT Assay buffer to the detection wells, and add 4 μL of the serum to be tested to the control wells. Mix well and incubate at 37°C for 60 min. After incubation, add 20 μL of ALT Assay buffer and 20 μL of dinitrophenylhydrazine color developing solution to the control wells, and add 20 μL of dinitrophenylhydrazine color developing solution to the detection wells. Mix well and incubate at 37°C for 20 min. After incubation, add 200 μL of ALT color developing base solution (1x) to each well. Mix well, place at room temperature for 5 min, and zero with distilled water. Measure the absorbance of each well at 505 nm using an enzyme label instrument.
[0048] 3.3.3 Determination of aspartate aminotransferase (AST) content
[0049] The AST detection kit was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd. (Batch number: S30HR196605).
[0050] Experimental procedure:
[0051] 3.3.1 Preparation of AST standard curve: Take one branch of pyruvic acid standard, accurately add 1 ml of pyruvic acid standard diluent, and mix well to prepare pyruvic acid standard (100 mmol / L) for 4°C storage. Before use, take an appropriate amount of pyruvic acid standard (100 mmol / L) and mix it according to the ratio of pyruvic acid standard (100 mmol / L): pyruvic acid standard diluent = 1:49 to obtain pyruvic acid standard working solution-pyruvic acid standard (2 mmol / L). Prepare a standard curve according to the AST Karmen enzyme activity units 0, 24, 61, 114, and 190 (KU). Set parallel detection wells and calculate the average value. Mix well, add 20 μL of dinitrophenylhydrazine color developing solution to each tube, incubate at 37°C for 20 min, then add 200 μL of AST color developing base solution (1x), mix well, and place at room temperature for 5 min. Zero with distilled water and measure the absorbance of each well at 505 nm using an enzyme label instrument. Subtract the absorbance of the "0" well from the absorbance of each well, and plot the difference in absorbance (ordinate) against the corresponding Karmen enzyme activity units (abscissa).
[0052] 3.3.2 AST enzymatic reaction: set control wells, assay wells, and detection wells. Add 4 μL of the serum to be tested and 20 μL of AST Assay buffer to the detection wells, and add 4 μL of the serum to be tested to the control wells. Mix well and incubate at 37°C for 60 min. After incubation, add 20 μL of AST Assay buffer and 20 μL of dinitrophenylhydrazine color developing solution to the control wells, and add 20 μL of dinitrophenylhydrazine color developing solution to the detection wells. Mix well and incubate at 37°C for 20 min. After incubation, add 200 μL of AST color developing base solution (1x) to the wells, mix well, and stand at room temperature for 5 min. Zero with distilled water, and measure the absorbance of the control wells and the assay wells at 505 nm using a microplate reader. Plot the standard curve using the standard activity units (24, 61, 114, and 190) as the abscissa and the corresponding absorbance as the ordinate. Subtract the absorbance of the control wells from the absorbance of the assay wells to obtain the difference, and then determine the AST activity units from the standard curve.
[0053] 3.3 Experimental results
[0054] Compared with the healthy control group, the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the serum of mice infected with E. multilocularis were significantly increased, indicating that E. multilocularis infection caused damage to the liver. Compared with the solvent control group, the alanine aminotransferase and aspartate aminotransferase contents in the serum of mice in the CA-170 treatment group were significantly reduced Figure 4 ). This result indicates that CA-170 treatment can effectively alleviate liver damage caused by E. multilocularis or alveolar echinococcosis infection.
Claims
1. Use of CA-170 in the manufacture of a medicament for treating multilocular echinococcosis, characterized in that, The structural formula of the CA-170 is shown as follows:
2. Use of CA-170 for the manufacture of a medicament for the treatment of multilocular echinococcosis according to claim 1, characterized in that, The hydatid disease is cystic development of the protocephalum caused by infection of Echinococcus multilocularis or liver damage caused by infection of Echinococcus multilocularis.
3. Use of CA-170 according to claim 1 or 2 for the manufacture of a medicament for the treatment of multilocular echinococcosis, characterized in that, The dosage form of the medicine is an oral dosage form.
4. Use of CA-170 according to claim 3 for the manufacture of a medicament for the treatment of multilocular echinococcosis, characterized in that, When CA-170 is administered, it is first dissolved in DMSO and then diluted with normal saline before administration.
5. Use of CA-170 according to claim 4 for the manufacture of a medicament for the treatment of multilocular echinococcosis, characterized in that, When CA-170 is administered, the dose of CA-170 in the medicine is not less than 10 mg / kg.
6. A pharmaceutical composition for treating multilocular echinococcosis, characterized by, The medicine comprises CA-170 or a pharmaceutically acceptable salt or hydrate of the salt thereof, and a pharmaceutically acceptable excipient.
7. A pharmaceutical composition for the treatment of hydatid disease according to claim 6, characterized in that, The dosage form of the medicine is an oral dosage form.
8. A pharmaceutical composition for the treatment of hydatid disease according to claim 7, characterized in that, The medicine is a tablet, granule, powder, capsule, oral liquid or buccal preparation.