Application of medicine for promoting hepatitis B virus clearance

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a method for promoting hepatitis B virus clearance by adjusting sex hormone level and application. According to the scheme, the androgen level is reduced, the serum HBsAg level is promoted to be reduced, and the serum HBsAg clearance rate is increased through a castration operation and a finasteride supplementing method; a male castrated hepatitis B mouse model test proves that in a castrated group, the negative conversion rate of serum HBsAg is about 42.86%; after a male hepatitis B mouse orally takes finasteride and the androgen is reduced, the serum HBsAg level of the male hepatitis B mouse is reduced, and the serum HBsAg negative conversion rate of 28.57% is obtained in the seventh week and is about 3 times that of a PBS oral group.

Description

Technical Field

[0001] The patent of this invention relates to the field of biomedicine technology, and specifically, to the application of a drug that promotes the clearance of hepatitis B virus. Background Art

[0002] Hepatitis B virus (HBV) is a DNA virus that primarily infects liver cells. HBV infection is the main pathogenic agent of cirrhosis and hepatocellular carcinoma. HBV infection and its complications are one of the seven leading causes of death worldwide, causing a heavy global public health burden. Globally, more than 2 billion people have been exposed to HBV, and as many as 316 million people have developed chronic hepatitis B (CHB). The annual incidence of CHB developing into cirrhosis is 2-10%, and after developing into cirrhosis, the annual probability of developing hepatocellular carcinoma is as high as 3-6%. Approximately 820,000 people die worldwide each year from cirrhosis, hepatocellular carcinoma, or liver failure caused by HBV infection. Clearance of hepatitis B surface antigen (HBsAg) (i.e., functional cure) can significantly reduce the incidence of cirrhosis and hepatocellular carcinoma and is associated with better clinical outcomes. However, under the natural infection state of HBV and existing treatment options, only a few people can achieve functional cure.

[0003] Existing treatment methods include two major categories, oral nucleoside (acid) analogs (Nucleoside / Nucleotideanalogue, NAs) and subcutaneous interferon drugs, both of which can inhibit HBV replication, reduce liver inflammation, fibrosis, and reduce the risk of cirrhosis and hepatocellular carcinoma, but rarely clear HBsAg and have many disadvantages. First, although NAs can effectively inhibit HBV replication, reduce viral load, delay the progression of CHB and significantly reduce the occurrence of end-stage liver disease, they cannot completely eliminate the risk of hepatocellular carcinoma; secondly, oral NAs rarely clear HBsAg (less than 1%), and viral rebound and recurrence are common after discontinuation of the drug. Therefore, oral NAs usually require lifelong treatment. However, long-term medication increases the risk of adverse reactions, leads to poor compliance, and thus affects its therapeutic effect and increases the risk of drug resistance. The cumulative incidence of drug resistance to entecavir is 1.2% in 5 years. Long-term use of tenofovir disoproxil fumarate poses a risk of renal damage and osteoporosis in high-risk groups. Alafenamide and tenofovir disoproxil fumarate may cause abnormal lipid metabolism. Although interferon α, mainly pegylated interferon alpha (Peg-IFN-α), can achieve clinical cure in some HBV-infected patients and reduce the incidence of HBV-related hepatocellular carcinoma, its subcutaneous injection treatment method, refrigerated storage method, multiple obvious side effects, and high cost have greatly limited its widespread application. In addition, Peg-IFN-α is contraindicated in patients with decompensated cirrhosis, pregnancy, severe cardiopulmonary complications, and immunosuppression. Therefore, it is urgent to explore and develop new anti-HBV infection strategies to pursue the transition from controlling viremia to achieving functional cure of CHB, and ultimately reduce the risk of cirrhosis and liver cancer in chronic HBV-infected patients.

[0004] After being infected with hepatitis B virus, the serum HBV replication and HBsAg levels in men are significantly higher than those in women. The number of men who develop chronic hepatitis is 2.4 times that of women, and the risk of male CHB patients developing hepatocellular carcinoma is 5-7 times that of women. The mechanism is closely related to the high androgen levels in the serum of male patients.

[0005] Finasteride, by inhibiting the activity of 5α-reductase in the body, reduces the level of dihydrotestosterone (an androgen with high biological activity). It is widely used clinically for male androgenic alopecia and benign prostatic hyperplasia. It has the characteristics of high safety, low price and easy administration.

[0006] To this end, the present application provides the use of lowering androgen levels and finasteride in the clearance of hepatitis B virus to increase the clearance rate of HBsAg and achieve a "functional cure" for hepatitis B. Summary of the invention

[0007] The purpose of the present invention is to solve the prior art problems raised in the above background technology and to provide a solution for optimizing the treatment of hepatitis B virus infection.

[0008] The above-mentioned purpose of the present invention is achieved like this: The scheme of the present invention provides the use of reducing androgen levels and finasteride in promoting HBsAg clearance.

[0009] The difficulty and significance of the technical problem solved by the present invention are: Higher androgen levels are closely related to the "three high phenomena" of high viral load, high HBsAg level, and high incidence of hepatocellular carcinoma in HBV-infected patients. Studies have shown that lowering androgen levels can reduce HBV viral replication, reduce serum HBsAg, and reduce the occurrence of hepatocellular carcinoma; conversely, after androgen supplementation, hepatitis B virus infection and complications are aggravated. At present, the development of new drugs for HBsAg clearance mainly focuses on drugs targeting the hepatitis B virus life cycle and drugs targeting host immune regulation. Although different androgen levels also have a great impact on the body's immune function, there are few reports on the value of regulating androgen levels in the treatment of hepatitis B. Therefore, the scheme of the present invention provides a new, safe, economical, and well-tolerated treatment for the current difficulties in the clinical treatment of chronic hepatitis B.

[0010] Compared with the prior art, the present invention has the following beneficial effects: 1. The androgen level reduction scheme provided by the present invention can promote the clearance of serum HBsAg, and experiments have confirmed that the average level of serum HBsAg in the castrated hepatitis B mouse model is 80% lower than that in the sham operation group, and the HBsAg negative conversion rate is about 42.86%, while there is no natural clearance of HBsAg in the control group; 2. The finasteride supplementation regimen provided by the present invention can promote the clearance of serum HBsAg. Experiments have confirmed that in the male hepatitis B mouse model supplemented with finasteride, the average serum HBsAg level was reduced by 42.86% compared with the PBS supplementation group. The HBsAg negative conversion rate reached 28.57% in only 7 weeks, which is about 3 times that of the oral PBS group. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : This is a diagram showing the effect of lowering androgen levels through male castration surgery to promote the clearance of serum HBsAg in a hepatitis B mouse model in an embodiment of the present invention ((a) serum HBsAg baseline value of a male castrated hepatitis B mouse model, (b, c) dynamic monitoring of serum HBsAg levels during the test, and (d) serum HBsAg negative conversion during the test); Figure 2This is a diagram showing the effect of reducing androgen levels by oral administration of finasteride to promote the clearance of serum HBsAg in a hepatitis B mouse model in an embodiment of the present invention ((a) baseline value of serum HBsAg in a hepatitis B mouse model after oral administration of finasteride, (b, c) dynamic monitoring of serum HBsAg levels during the test, and (d) negative conversion of serum HBsAg during the test). DETAILED DESCRIPTION

[0012] In order to more clearly demonstrate the purpose, technical solutions and advantages of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0013] The implementation of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0014] The following is a specific test of the embodiment of the present invention: Experimental Example 1: Investigation of the effect of reducing androgen levels on HBV clearance by castration of male mice according to the present invention Experimental methods After castration surgery was performed on male mice, pAAV / HBV1.2 plasmid was injected into the tail by intravenous hydrodynamic high pressure to construct an HBV mouse model, and the effect of reducing androgen levels on HBV clearance by the present invention was investigated by detecting serum HBsAg levels.

[0015] (1) Surgical operation of male mice: After 1 week of adaptive feeding, 4-week-old C3H / HeN male mice were randomly divided into 2 groups and underwent castration surgery and sham surgery, respectively. Castration surgery: After the mice were anesthetized, they were fixed on the operating table in a supine position with their limbs. The skin under the abdomen was disinfected with 70% alcohol cotton balls, the hair was shaved, the testicle was pushed down as much as possible, and a 1 cm longitudinal incision was made in the midline of the testicle with surgical scissors. The subcutaneous tissue was separated with forceps to expose the spermatic cord sac for easy separation. Small curved forceps were used to explore and bluntly separate the testicular sac on one side of the spermatic cord to the bottom of the scrotum, and the testicular sac was pulled out to expose the testicle and the ipsilateral epididymis. The testicle, epididymis and surrounding tissues were gently clamped out, the testicular artery and spermatic artery were separated and ligated, the spermatic cord was ligated, and then the testicle and epididymis were removed. The same operation was performed on the contralateral side, and the muscle layer and skin were sutured with 6-0 and 4-0 absorbable surgical sutures, respectively. Sham operation: After anesthesia, incision, and removal of the testicle and epididymis, only the bilateral testicular arteries and spermatic arteries were separated without ligating the arteries or removing the testicles. The testicles and epididymis were returned to the scrotum and sutured. The mice rested for 1 week before the next experiment.

[0016] (2) Injection of recombinant plasmid: Dissolve 10 μg of pAAV / HBV 1.2 recombinant plasmid in PBS equivalent to 8% of the mouse body weight, mix well and transfer to a syringe for use. Inject the HBV recombinant plasmid into the terminal 1 / 3 of the mouse tail vein, and complete the injection within 5 to 8 seconds. After 24 hours, detect HBsAg in the mouse peripheral blood serum to confirm that the hepatitis B mouse model was successfully established.

[0017] Subsequently, mouse serum was collected weekly and serum HBsAg was tested until it became negative (<0.05 IU / mL) or until the observation endpoint (24 weeks).

[0018] (3) Serum collection: After isoflurane inhalation anesthesia, about 150 µL of blood was collected from the inner canthus of the mouse and placed in a 1.5 m EP tube. The blood was allowed to stand at room temperature for 1 hour and centrifuged at 3000 rpm for 15 minutes. The upper layer of serum was aspirated into a new EP tube and stored in a -80°C medical refrigerator to prevent repeated freezing and thawing of the serum.

[0019] (4) Detection of serum HBsAg: The Abbott ARCHITECT instrument was used to quantitatively detect the HBsAg level in mouse serum collected weekly.

[0020] Experimental Results Detection results of serum HBsAg levels in hepatitis B mice and serum HBsAg clearance Figure 1 a is the baseline value of serum HBsAg in hepatitis B mice, Figure 1 b is the comparison of serum HBsAg in the two groups of mice at the 10th week. Figure 1 c shows the dynamic monitoring of serum HBsAg levels. Figure 1 d shows the situation of serum HBsAg negative conversion (HBsAg < 0.05 IU / mL).

[0021] It can be seen from the above test results that the HBsAg level decreased significantly after the androgen level was lowered by castration of male mice. This effect became more significant and persisted as the androgen level decreased, and low androgen levels could significantly promote the clearance of HBsAg.

[0022] Experimental Example 2: Investigation of the effect of reducing androgen levels by supplementing finasteride on HBV clearance in the present invention Experimental methods After 1 week of adaptive feeding, 5-week-old C3H / HeN male mice were hydrodynamically injected with 10 μg pAAV-HBV1.2 plasmid into the tail vein according to the above method to construct the hepatitis B model. The mice with successful modeling were randomly divided into two groups, the finasteride gavage group and the PBS gavage group. Then, according to the above method, the HBsAg level in the mouse serum was detected every week until it turned negative or the observation endpoint.

[0023] Experimental Results The baseline value of serum HBsAg in hepatitis B mice is as follows Figure 2 As shown in a, Figure 2 b is the comparison of serum HBsAg levels of the two groups of mice at week 7. Figure 2 c shows the dynamic monitoring of serum HBsAg levels. Figure 2 d shows the negative conversion of serum HBsAg.

[0024] It can be seen from the above test results that lowering androgen levels through finasteride can significantly reduce HBsAg levels and promote HBsAg clearance.

[0025] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of lowering androgen levels in promoting the clearance of hepatitis B virus.

2. The application of finasteride in promoting the clearance of hepatitis B virus by reducing androgen levels.

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