Use of fingolimod in preparing a drug for inhibiting hepatitis B virus replication

Abstract

The present invention provides use of fingolimod in a free form or a pharmaceutically acceptable salt form in the preparation of a drug for preventing or treating a disease induced by hepatitis B virus.

Description

Technical Field

[0001] The present application relates to the field of medicine, and specifically to a new use of fingolimod in inhibiting hepatitis B virus replication and hepatitis B liver damage. Background Art

[0002] Fingolimod hydrochloride (FTY720), also known as Fingolimod hydrochloride in Chinese, is a sphingosine analog and an effective sphingosine 1-phosphate (S1P) receptor modulator. Fingolimod hydrochloride is phosphorylated by sphingosine kinase, especially by SK2, and can then bind to S1PR1, 3, 4, and 5. Fingolimod hydrochloride induces the internalization of S1P1 and therefore has the activity of inhibiting S1P.

[0003] The molecular weight of FTY720 is 343.93 and the molecular formula is C 19 H 34 ClNO 2 , the chemical structure is:

[0004]

[0005] The main clinical application direction of FTY720 is immunosuppressant. FTY720 is a new type of immunosuppressant derived from the ascomycete Cordyceps sinensis. Compared with conventional immunosuppressants such as cyclosporine, tacrolimus, and sirolimus, it has a different mechanism of action. It mainly exerts immunosuppressive effects by inducing lymphocyte apoptosis and homing effects. It has the effect of inhibiting graft rejection in allogeneic transplantation in various animal models, and also shows strong immunosuppressive activity in human kidney transplantation. Therefore, it has broad application prospects in clinical organ transplantation and the treatment of autoimmune diseases.

[0006] Approved clinical applications include: Novartis's Fingolimod has been listed as the first batch of overseas new drugs urgently needed in clinical practice by the Center for Drug Evaluation (CDE) of the National Medical Products Administration, and was approved for marketing in China in 2019, mainly for the treatment of multiple sclerosis (the first oral medication). Since its launch in 2010, Fingolimod's sales have continued to grow. In view of the adverse reactions of Fingolimod, Novartis has developed a second-generation MS drug Siponimod, which is a new generation of selective S1PR modulators and has been included in the priority review list by my country's CDE.

[0007] CN106667981A discloses the use of fingolimod hydrochloride (FTY720) in the preparation of drugs for treating drug-induced liver injury, especially ... in the preparation of drugs for treating drug-induced liver injury, such as reducing the levels of alanine aminotransferase and aspartate aminotransferase in liver injury induced by acetaminophen (APAP), reducing the area of ​​liver tissue necrosis, reducing the mortality of mice induced by lethal doses of APAP, and reducing the expression of inflammatory factors and chemokines in drug-induced liver injury.

[0008] There is no report on the use of FTY720 in the treatment of hepatitis B in the prior art. The main difficulty in the treatment of hepatitis B is that direct antiviral drugs such as entecavir can inhibit the replication of hepatitis B virus, but cannot eliminate cccDNA in hepatitis B virus HBV cells. Using cccDNA as a template, some important functional regulatory proteins of the virus such as HBx will be synthesized. HBx can regulate the expression of some genes in liver cells, affecting the proliferation, apoptosis, lipid metabolism, sugar metabolism, etc. of liver cells. These changes in liver cells induce some autoimmune reactions, making the liver microenvironment in a state of immune damage for a long time, and then develop into hepatitis B-related liver diseases such as liver fibrosis, liver failure, cirrhosis, and cancer.

[0009] The present invention found that FTY720 can not only directly inhibit viral replication, but also inhibit the transcription and translation of HBx, reduce the synthesis of virus-related regulatory proteins that cause liver damage, and also inhibit the activation and chemotaxis of T cells. Summary of the invention

[0010] The present invention provides use of fingolimod in a free form or a pharmaceutically acceptable salt form in the preparation of a drug for preventing or treating a disease induced by hepatitis B virus.

[0011] In certain embodiments, the disease comprises hepatitis B virus-induced hepatocellular damage, necrosis or abnormal liver function.

[0012] In certain embodiments, the disease includes hepatitis B and hepatitis B virus-induced liver fibrosis, liver failure, cirrhosis or cancer.

[0013] In certain embodiments, the disease comprises acute hepatitis B, chronic hepatitis B, severe hepatitis B (liver failure), cholestatic hepatitis B (capillary cholangitis hepatitis B), hepatitis B cirrhosis, hepatitis B liver fibrosis, and hepatocellular carcinoma (HCC) associated with hepatitis B.

[0014] In certain embodiments, the pharmaceutically acceptable salt form of fingolimod is fingolimod hydrochloride.

[0015] The present invention further provides a pharmaceutical composition comprising

[0016] a) a first component, which is fingolimod in free form or in a pharmaceutically acceptable salt form as described above, and

[0017] b) The second component is an auxiliary agent having anti-HBV activity.

[0018] In certain embodiments, the second component is selected from one or more of entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide.

[0019] Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0021] Fig. 1 FTY720 inhibits chemotaxis of immune cells induced by HBV infection Implementation

[0022] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0023] Example 1: FTY 720 inhibits viral replication and HBx synthesis

[0024] 1. Experimental process

[0025] (1) Hepatitis B cancer cell line HepG2.2.15 model

[0026] HepG2.2.15 is a liver cancer cell line expressing HBV virus and is often used for screening of hepatitis B related drugs. The specific experimental steps are as follows.

[0027] ①HepG2.2.15 cells were cultured in MEM complete medium containing 10% FBS. 5 Each well was inoculated in a 12-well plate.

[0028] ② 1.5ug / ml of entecavir (ETV, a drug currently commonly used in clinical treatment of hepatitis B) and 5uM FTY720 were added to the culture medium of each well, and untreated cells were used as controls.

[0029] ③ After 2 days of co-culture, the supernatant was collected to detect related viral markers, and the cells were collected to detect intracellular viral markers.

[0030] (2) HBV virus infection HepG2-NTCP model

[0031] HepG2-NTCP is a liver cancer cell that overexpresses the hepatitis B virus receptor NTCP. It does not contain hepatitis B virus itself. Exogenous HBV virus particles can be used to infect HepG2-NTCP cells to simulate the process of viral infection in vitro. It is a commonly used HBV infection model and can be used for experimental research in drug screening, research on the mechanism of hepatitis B infection, etc.

[0032] ① Exogenous hepatitis B virus, derived from HBsAg positive and HBV DNA greater than 10 7 IU / ml of patient serum, and the negative control was HBV-negative healthy human serum, which was sterilized by filtration before infecting cells.

[0033] ②HepG2-NTCP cells were cultured in DMEM complete medium containing 10% FBS for 10 6 Each well of the cells were inoculated into a 6-well plate and allowed to adhere to the plate for more than 6 hours.

[0034] ③HBV virus contact: add "500ul of virus-containing positive serum + 2% polyethylene glycol 8000 (PEG8000, concentrated aggregated virus) + 1% DMSO" to a total volume of 2ml of culture medium and co-culture with the above-mentioned adherent cells overnight. Negative serum without HBV virus was treated synchronously as a negative control; culture wells without human serum were treated synchronously as blank controls.

[0035] ④ Residual HBV virus removal: discard the culture supernatant after overnight and wash with PBS for more than 6 times to remove residual virus.

[0036] ⑤HBV virus infection: add fresh culture medium, fresh culture medium + 1.5ug / ml entecavir (ETV), fresh culture medium + 5uM FTY720, and continue culturing.

[0037] ⑥ After 2 days of culture, collect the supernatant to detect related viral markers, and collect cells to detect intracellular viral markers.

[0038] (3) Detection of HBV virus-related markers

[0039] ① The culture supernatant obtained in the above model was used to quantitatively detect HBsAg and HBeAg, commonly used hepatitis B serum antigen markers, using the Abbott instrument using chemiluminescence method.

[0040] ② For intracellular HBx detection, total RNA from cultured cells was extracted manually using the Trizol method and reverse transcribed into cDNA (Novagen reverse transcription kit). β-ACTIN (intracellular reference) and HBx (Novagen quantitative PCR kit) were detected by qPCR, and the relative changes in HBx mRNA expression levels were determined by the 2-ΔΔct method.

[0041] ③ Intracellular HBV cccDNA detection: Cell lysis buffer (ShuoShi) was used to release cellular nucleic acid in one step, and the HBV cccDNA quantitative detection kit used qPCR to detect the cccDNA content. Based on β-ACTIN (cellular internal reference), the 2-ΔΔct method was used to determine the relative changes in HBV cccDNA expression levels.

[0042] 2. Experimental results

[0043] (1) Cell line G2.2.15 model results

[0044] The experimental results of the cell line G2.2.15 model are shown in Table 1:

[0045]

[0046] (2) Results of HepG2-NTCP infection model

[0047] The experimental results of HepG2-NTCP infection model are shown in Table 2:

[0048]

[0049] Note: HBsAg: hepatitis B surface antigen; HBeAg: hepatitis B e antigen; HBV DNA: hepatitis B virus deoxyribonucleic acid; HBx: hepatitis B virus x protein; cccDNA: hepatitis B virus covalently closed circular DNA.

[0050] Experiments show that:

[0051] (1) After FTY720 intervention, the viral load was significantly reduced, indicating that it has a direct inhibitory effect on HBV replication. In the HepG2-NTCP infection model, the inhibitory effect on HBV was slightly stronger than that of entecavir; in the cell line G2.2.15 model, the inhibitory effect on HBV was slightly weaker than that of entecavir; overall, FTY720 and entecavir both have a direct inhibitory effect on HBV replication, and the effects are comparable.

[0052] (2) After intervention with FTY720, it has a significant clearance effect on HBV cccDNA and a significant inhibitory effect on the synthesis of viral protein HBx, with a significantly stronger effect than entecavir. Existing hepatitis B treatment drugs can only achieve functional cure in very few patients with chronic hepatitis B (CHB). Even with the most effective combination therapy, the annual negative conversion rate of hepatitis B surface antigen is difficult to exceed 2%. The key to achieving CHB cure lies in effectively inhibiting or clearing the highly stable genetic material formed by HBV in cells - minichromosome-like covalently closed circular DNA (cccDNA). The viral protein HBx plays a key role in initiating and maintaining cccDNA transcription. Therefore, targeting the HBx protein is an important means to achieve cccDNA transcription shutdown. HBx binds to other proteins in the cell and can also control the transcription and translation of other genes in the cell. It is a key functional protein of HBV.

[0053] Example 2: FTY 720 inhibits immune cells

[0054] 1. FTY720 inhibits chemotaxis of immune cells induced by HBV infection

[0055] (1) Test process

[0056] ① Pre-dissolve one Matrigel (Corning) on ​​ice at 4°C

[0057] ② Prepare Matrigel: serum-free medium = 1:8 dilution, add 100ul to the upper chamber surface of the chamber, place in a 37° incubator for 4 hours to allow Matrigel to polymerize into gel, and be careful to gently aspirate the unsolidified liquid on the upper layer

[0058] ③ Preparation of PBMC cell suspension: Draw 10 ml of whole blood, separate peripheral blood mononuclear cells, wash twice with sterile PBS, and make 2 ml suspension. Take 200 ul and count 10×10 6 / mL, dilute in multiple ratios, take out 1ml of the suspension and add 1ml of serum-free medium, mix well, and reach 5×10 6 / mL, add 200ul to each chamber

[0059] ④ Preparation of chemotactic factors in the lower chamber: The chemotactic factors were divided into three groups: a. Cell culture supernatant of HepG2-NTCP cells infected with HBV negative normal human serum at 0 days, 3 days, 6 days and 9 days; b. Cell culture supernatant of HepG2-NTCP cells infected with HBV positive patient serum at 0 days, 3 days, 6 days and 9 days; c. 5uMFTY720 was added to the culture supernatant of the hepatitis B positive group.

[0060] ⑤ Add 600ul of culture medium containing corresponding chemotactic factors into the 24-well plate (lower chamber): 200ul of fresh DMEM complete culture medium containing 10% FBS + 400ul of chemotactic factors prepared in ④ + 8ul of double antibody, and move the prepared chamber to the middle two rows.

[0061] ⑥ Add 200ul PBMC cell suspension to each well of the chamber.

[0062] ⑦After 12 hours, fix the cells: remove the Transwell chamber, aspirate the liquid in the chamber, wash twice with 800ul PBS, and gently wipe the Matrigel and cells in the upper chamber with a cotton swab; add 600µL of 4% paraformaldehyde to the clean wells of the 24-well plate, put the chamber in and fix for 30 minutes.

[0063] ⑧Cell staining and counting: discard the fixative, wash the chamber with PBS; stain with 0.1% crystal violet for 5-10 minutes, rinse 3 times with PBS; count after air drying.

[0064] ⑨ Counting: Take out the chamber, place it on a clean glass slide, observe the cells in five random fields under a 400x microscope, count them, and record the number of each chamber.

[0065] (2) Experimental results

[0066] The experimental results are as follows Figure 1 As shown, it can be seen that the culture products after HBV infection of hepatocytes induced a lot of T cell chemotaxis. After adding FTY720, the number of mononuclear cells chemotactic to the HBV infection chamber was significantly reduced.

[0067] 2. FTY720 inhibits the activation of immune cells caused by HBV infection

[0068] ① Preparation of HBV infected cells: See Example 1.1 (2) for the experimental method, wherein HepG2-NTCP cells were infected with HBV negative normal human serum and cultured for 6 days (negative uninfected cells), and HepG2-NTCP cells were infected with HBV positive patient serum and cultured for 6 days (HBV infected cells).

[0069] ② Preparation of T cells: Peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteers, and CD3+ T cells were sorted by flow cytometry.

[0070] ③Cell co-culture: The above two cells were co-cultured at a ratio of hepatocytes: T cells = 1:2 and divided into three groups: negative uninfected co-culture group, HBV-infected cell co-culture group, HBV-infected cell + 5uM FTY720 inhibitor group + T cell co-culture group.

[0071] ④After co-culture for 48 hours, aspirate the supernatant containing the suspended T cells and wash with PBS for subsequent detection.

[0072] ⑤ Detect the number of CD3+CD4+ and CD3+CD8+ cells by flow cytometry, and calculate the percentage and CD4+ / CD8+ ratio.

[0073] (2) Results

[0074] The experimental results are shown in Table 3. It can be seen that when HBV-infected hepatocytes and T cells were co-cultured, FTY720 significantly inhibited the activation of T cells towards CD4+T (helper T cells).

[0075]

[0076] 3. Conclusion

[0077] After HBV infects liver cells, it will chemotaxis T cells and other immune cells to the infection site, causing local immune damage to the liver and a series of lesions such as fibrosis. Therefore, inhibiting the abnormal chemotaxis and activation of immune cells is also one of the important mechanisms to inhibit immune liver damage caused by HBV infection. FTY20 can significantly inhibit the abnormal chemotaxis and activation of immune cells, thereby achieving the effect of inhibiting immune liver damage caused by HBV infection.

[0078] Example 3: Evaluation of the inhibitory effect of FTY20 on liver function damage

[0079] FTY720 was added to the hepatitis B infection hepatocyte test, and compared with the control group without FTY720, to compare whether the concentrations of liver damage markers such as ALT and AST, which are indicators of liver function, were reduced.

[0080] 1. Experimental process

[0081] (1) Hepatitis B cancer cell line HepG2.2.15 model

[0082] ①HepG2.2.15 cells were cultured in MEM complete medium containing 10% FBS. 6 Each well was inoculated in a 12-well plate.

[0083] ② 1.5ug / ml of entecavir (ETV, a drug currently commonly used in clinical treatment of hepatitis B) and 5uM FTY720 were added to the culture medium of each well, and untreated cells were used as controls.

[0084] ③After 3 days of co-culture, the supernatant was collected to detect liver function indicators.

[0085] 2. Results

[0086] The results are shown in Table 4:

[0087]

[0088] (Note: ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphatase; PA: prealbumin; ALB: albumin; TP: total protein.

[0089] From the above results, it can be seen that FTY720 has a significant improvement effect on some liver function indicators, mainly including aspartate aminotransferase (AST), alkaline phosphatase (ALP), prealbumin (PA), and albumin (ALB), a total of 4 items.

Claims

1. Use of fingolimod in the preparation of a medicament for preventing or treating hepatitis B, characterized in that: The fingolimod is in free form or a pharmaceutically acceptable salt.

2. The use according to claim 1, characterized in that The diseases include acute hepatitis B, chronic hepatitis B, severe hepatitis B, and cholestatic hepatitis B.

3. The use according to any one of claims 1 to 2, characterized in that The pharmaceutically acceptable salt form of fingolimod is fingolimod hydrochloride.

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