Application of fatty acid amide hydrolase inhibitor PF-3845 in the preparation of drugs for treating alveolar echinococcosis

Abstract

The present invention discloses the use of the fatty acid amide hydrolase inhibitor PF-3845 as a drug for the preparation of a drug for treating alveolar echinococcosis. The present invention finds that the fatty acid amide hydrolase inhibitor PF-3845 can destroy the head hook, sucker, microvilli and other structures of the protoscolecus of Echinococcus multilocularis, causing the protoscolecus to lyse and die, and can also destroy the structure of the vesicles in the tapeworm stage, causing the vesicles to collapse. This shows that the fatty acid amide hydrolase inhibitor PF-3845 has a good killing effect on Echinococcus multilocularis and can be used as an alternative to albendazole to treat echinococcosis, with good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine and application of a fatty acid amide hydrolase inhibitor PF-3845 as a medicine for preparing a medicine for treating alveolar echinococcosis. Background Art

[0002] Alveolar echinococcosis (AE) is caused by Echinococcus multilocularis ( Echinococcusmultilocularis , E. m ) larvae (alveolar cysts) parasitize the human liver and cause a fatal parasitic disease. Clinically, the disease presents similarly to liver cancer, often referred to as "worm cancer." AE poses a serious threat to public health and safety, becoming a major public health concern and one of the "new five parasitic diseases" requiring priority prevention and treatment. Currently, surgical resection of the lesion is the primary treatment for AE. For patients who are unable to undergo surgery or who need to prevent recurrence after surgery, drug therapy is the preferred treatment. The WHO recommends albendazole (ABZ) as the preferred treatment for echinococcosis. However, ABZ has low intestinal absorption and must be converted to albendazole sulfoxide in the liver to be effective. Albendazole sulfoxide kills echinococcosis primarily by interfering with its energy metabolism and affecting the structure and function of microtubules. This has significant side effects on the liver, resulting in low bioavailability and poor efficacy of ABZ. Therefore, the development of effective treatments for AE is urgently needed. In recent years, research and application of new drug therapies for echinococcosis have focused on new drug discovery.

[0003] Fatty acid amide hydrolase (FAAH), a member of the serine hydrolase family, is one of the primary hydrolases of endogenous cannabinoids. Widely present in the nervous system, FAAH regulates the degradation of N-arachidonic acid ethanolamine, affecting cell proliferation, apoptosis, and differentiation. It also participates in regulating various physiological functions, including pain and appetite, neurotransmitter release, and inflammatory responses. Studies have reported that FAAH inhibitors can indirectly increase N-arachidonic acid ethanolamine levels in the body and exert anti-tumor effects by inducing apoptosis and inhibiting tumor cell migration and angiogenesis.

[0004] PF-3845 is a highly potent, selective, irreversible, and orally available FAAH inhibitor that covalently inhibits FAAH activity by reacting with a serine nucleophile in a carbamate reaction. In vivo studies have shown that the FAAH inhibitor PF-3845 can significantly reduce pain, inflammation, and fight tumors in a variety of diseases. However, there have been no reports of the use of the FAAH inhibitor PF-3845 for the treatment of alveolar echinococcosis. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to treat and / or prevent alveolar echinococcosis.

[0006] To solve the above technical problems, the present invention first provides any of the following applications:

[0007] 1. Use of a substance that inhibits FAAH gene expression in the preparation of a product for treating and / or preventing alveolar echinococcosis, or in the treatment of alveolar echinococcosis;

[0008] 2. Use of substances that reduce the content or activity of FAAH in the preparation of products for treating alveolar echinococcosis, or in the treatment of alveolar echinococcosis;

[0009] 3. Use of FAAH inhibitors in the preparation of products for treating alveolar echinococcosis, or in the treatment of alveolar echinococcosis;

[0010] 4. Use of substances that inhibit FAAH gene expression in the preparation of products for reducing the activity of Echinococcus multilocularis, or in reducing the activity of Echinococcus multilocularis;

[0011] 5. Use of a substance that reduces FAAH content or activity in the preparation of a product for reducing the activity of Echinococcus multilocularis, or in reducing the activity of Echinococcus multilocularis.

[0012] 6. Use of a FAAH inhibitor in the preparation of a product for reducing the activity of Echinococcus multilocularis, or in reducing the activity of Echinococcus multilocularis.

[0013] In the above, the FAAH inhibitor may be PF-3845.

[0014] The treatment and / or prevention of alveolar echinococcosis can be reflected in the killing of Echinococcus multilocularis.

[0015] The multilocular Echinococcus tapeworm may be a protoscolecus or a tapeworm stage vesicle (a multilocular Echinococcus tapeworm larva: i.e., an echinococcosis, also known as a tapeworm stage).

[0016] In the present invention, the product may be a medicine.

[0017] Experiments have shown that the fatty acid amide hydrolase inhibitor PF-3845 can destroy the head hook, sucker, microvilli, and other structures of the protoscolecus of Echinococcus multilocularis, causing the protoscolecus to lyse and die. It can also destroy the structure of the vesicles in the tapeworm stage, causing vesicle collapse. This indicates that the fatty acid amide hydrolase inhibitor PF-3845 has a strong killing effect on Echinococcus multilocularis and can be used as a drug to treat alveolar echinococcosis.

[0018] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :The effects of different concentrations of FAAH inhibitor PF-3845 on the killing of Echinococcus multilocularis protoscoleces (methylene blue staining).

[0020] Figure 2 :The effects of different concentrations of FAAH inhibitor PF-3845 on the survival rate of Echinococcus multilocularis protoscolex.

[0021] Figure 3 :The effects of FAAH inhibitor PF-3845 (20 μM) on the morphology of the protoscolecus of Echinococcus multilocularis were observed under optical microscopy.

[0022] Figure 4 :The effects of FAAH inhibitor PF-3845 (20 μM) on the morphology and structure of the protoscolecus of Echinococcus multilocularis were observed using scanning electron microscopy.

[0023] Figure 5 :The effects of lipid FAAH inhibitor PF-3845 (20 μM) on the morphology and structure of vesicles in the tapeworm stage of Echinococcus multilocularis were observed under optical microscopy.

[0024] Figure 6 :The effects of FAAH inhibitor PF-3845 (20 μM) on the morphology and structure of vesicles in the tapeworm stage of Echinococcus multilocularis were observed using scanning electron microscopy. DETAILED DESCRIPTION

[0025] Unless otherwise noted, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in literature in the field or according to product specifications. The materials, reagents, and instruments used in the following examples are all commercially available unless otherwise noted. The quantitative experiments in the following examples were performed in at least three replicates, and the results were averaged.

[0026] PF-3845: Abcam; Catalog Number: AB120894; CAS: 1196109-52-0.

[0027] Example 1: Application of the fatty acid amide hydrolase inhibitor PF-3845 in the preparation of a drug for treating alveolar echinococcosis

[0028] This example obtains data on the killing of the fatty acid amide hydrolase inhibitor PF-3845 on the protoscoleces and vesicles of Echinococcus multilocularis cultured in vitro, and finds that the fatty acid amide hydrolase inhibitor PF-3845 can be used to prepare a drug for treating alveolar echinococcosis. The application test is as follows:

[0029] 1. Drug intervention of Echinococcus multilocularis protoscoleces cultured in vitro

[0030] The protoscoleces (i.e., larval stage) of Echinococcus multilocularis were aseptically isolated in vitro and placed in a 96-well plate containing 200 μL of culture medium (1% penicillin (mass percentage) + 10% fetal bovine serum (volume percentage) + RPMI-1640 medium). Then, different concentrations of the fatty acid amide hydrolase inhibitor PF-3845 were added to the culture system for intervention. PF-3845 was first dissolved in DMSO to prepare a 200 mM stock solution. The experiment set up PF-3845 intervention groups (1 μM, 10 μM, 25 μM, and 50 μM, that is, the final concentrations of PF-3845 in the culture system were set to 1 μM, 10 μM, 25 μM, and 50 μM, respectively) and a solvent control group (final concentration was 0.1% DMSO control group, that is, DMSO of the same volume as the intervention group was added to the culture system, with a final concentration of 0.1%). The worms were collected 3 days, 6 days, 9 days, and 12 days after the intervention, and the activity of the protoscolex was detected by methylene blue staining. The morphological and structural changes of the worms 12 days after the intervention were observed by scanning electron microscopy.

[0031] The experimental results are shown in Figure 1 and Figure 2 After 12 days of intervention, the protoscolex activity of the 0.1% DMSO control group was 93 ± 0.7%, and the protoscolex activity of the PF-3845 intervention groups at 1 μM, 10 μM, 25 μM, and 50 μM decreased to 93 ± 3.1%, 43 ± 8.5%, 3 ± 1.0%, and 2 ± 1.4%, respectively. The protoscolex activity of PF-3845 at concentrations of 25 μM and 50 μM was significantly lower than that of the 0.1% DMSO control group.

[0032] Following the same drug intervention method as above, the final concentration of PF-3845 was set to 20 μM. The results showed that after 12 days of treatment with the inhibitor PF-3845 at a concentration of 20 μM, optical microscopy revealed that the activity of the protoscolecus decreased, the body atrophied, and the head hook fell off ( Figure 3 Scanning electron microscopy revealed that the protoscolex was atrophied, the head hook had fallen off, the sucker was destroyed, and the microvilli were truncated ( Figure 4 ). This indicates that the fatty acid amide hydrolase inhibitor PF-3845 has a good killing effect on the protoscoleces of Echinococcus multilocularis cultured in vitro.

[0033] 2. Drug intervention of Echinococcus multilocularis tapeworm vesicles cultured in vitro

[0034] Vesicles of the tapetum stage of Echinococcus multilocularis were isolated by aseptic culture in vitro and plated in 24-well plates containing 1 mL of culture medium (1% penicillin-streptomycin, 10% fetal bovine serum, and RPMI-1640). The culture medium was then treated with varying concentrations of the fatty acid amide hydrolase inhibitor PF-3845, which was dissolved in DMSO to a 200 mM stock solution. A PF-3845 treatment group (20 μM, final concentration of PF-3845 in the culture system) and a solvent control group (0.1% DMSO, equivalent volume of DMSO to that in the treatment group, final concentration of 0.1% (volume percentage)) were also included. The integrity and collapse of the tapetum stage vesicles were observed by light microscopy and electron microscopy 12 days after treatment.

[0035] The experimental results are shown in Figure 5 and Figure 6 In the intervention group, PF-3845 (20 μM), a fatty acid amide hydrolase inhibitor, was used to treat E. multilocularis vesicles for 12 days. The vesicle walls lost their luster and showed obvious collapse. This suggests that PF-3845 has a good killing effect on E. multilocularis vesicles cultured in vitro.

[0036] In summary, the present invention discloses for the first time that fatty acid amide hydrolase inhibitor PF-3845 is a new type of anti-alveolar echinococcosis drug; pharmacodynamic experimental data show that fatty acid amide hydrolase inhibitor PF-3845 has a killing effect on the protoscolecus of Echinococcus multilocularis and inhibits the growth of vesicles in the tapeworm stage, indicating that PF-3845 can be used to treat alveolar echinococcosis.

[0037] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Application of PF-3845 in the preparation of products for the treatment and / or prevention of alveolar echinococcosis.

2. The use according to claim 1, characterized in that: The treatment and / or prevention of alveolar echinococcosis is embodied in the killing of the protoscoleces or vesicles of Echinococcus multilocularis.

3. Application of PF-3845 in the preparation of products for reducing the activity of Echinococcus multilocularis.

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