CN1775217A - Pharmaceutical composition for treating hepatitis B - Google Patents

Abstract

The pharmaceutical composition for treating hepatitis B, which contains the compound of formula (I) Alisol A (Alisol A) as an active ingredient, and contains conventional pharmaceutical carriers. Application of the above-mentioned drug compound alisol A (Alisol A) in the preparation of anti-hepatitis B medicine and anti-hepatitis B inhibitor medicine.

Description

Pharmaceutical composition for treating hepatitis B

Field of the invention: the present invention belongs to the technical field of medicines, and in particular, relates to a pharmaceutical composition for treating hepatitis B and its application in the preparation of anti-hepatitis B inhibitor drugs and in the preparation of anti-hepatitis B drugs.

BACKGROUND OF THE INVENTION: Hepatitis, which is commonly referred to by people, is viral hepatitis. Hepatitis caused by seven hepatitis viruses has been found to be hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G and TTV. Among these seven kinds of viral hepatitis, hepatitis A and E are transmitted through the gastrointestinal tract and are self-limiting diseases that will not develop into chronic hepatitis. Hepatitis D virus is a defective virus that can only replicate if attached to hepatitis B virus; hepatitis G does not cause widespread transmission. Hepatitis B and C are viral infectious diseases that seriously endanger human health. Hepatitis B virus belongs to DNA Hepadnaviridae, and Hepatitis C virus belongs to RNA Flaviviridae. Its transmission route is through blood, mother-infant and sexual contact, etc., and it is easy to turn into chronic hepatitis, which is the main cause of liver cirrhosis and liver cancer. . The replication process of HBV involves 5 steps: binding, invasion, transcription, translation, gene replication and packaging. First, the granules attach to the liver cells through receptors, invade the cytoplasm and remove the outer membrane to form core granules. The core particle moves to the nucleus of the liver cell, and the right core particle of HBV DNA is shelled out, forming cccDNA in the nucleus. cccDNA is the membrane plate for the replication of hepatitis B virus, and is the key substance for the long-term persistence of infection. cccDNA has two functions, one is to synthesize various components of virus particles, such as HBsAg (surface antigen, constituting the outer membrane), HBcAg (core antigen, constituting the inner membrane), HBeAg (e antigen, secreted into the blood); The second is to synthesize the next generation of HBV DNA. Most of the newly synthesized HBV DNA is used for the packaging of new virus particles, and part of it re-enters the liver cell nucleus and becomes another source of cccDNA. Part of the newly synthesized HBV DNA first assembles with HBcAg to form a core particle, and then assembles with HBsAg to form a complete HBV particle, which is released outside the cell. HBsAg is sometimes excessive and released into the blood alone, forming small spherical particles or cast particles.

At present, there is still a lack of specific therapeutic drugs for the treatment of viral hepatitis B and C. The development of new drugs for the treatment of viral hepatitis in foreign countries focuses on the development of anti-hepatitis virus drugs, and some progress has been made in the research of anti-hepatitis virus chemotherapy drugs. Adenosine vidarabine, adenosine vidarabine phosphate, acyclovir, and zidovudine, which were tested in the 1980s, are no longer used to treat hepatitis B abroad because of their poor curative effect and high toxicity. In recent years, a variety of nucleoside drugs have been developed, which have obvious inhibitory effects on HBV. Lamivudine, famciclovir, lobucavir, adefovir dipivoxiil, FTC (dideoxyfluorothiocytosine), FMAU (fluoromethyl-arabinouracil), FDDC (fluorodideoxycytosine), BMS 200475 (epoxy hydroxycarbodeoxyguanosine) These drugs have the following things in common: they are all nucleoside derivatives, which are cytosine, uracil, guanine or purine compounds. In in vitro or in vivo experiments, it has inhibitory effect on viruses such as HIV, HBV or HCV. Its mechanism of action is to enter the cell and become a triphosphate compound, through the competition of the substrate, inhibit the polymerase or reverse transcriptase of the virus, and finally inhibit the synthesis of viral DNA and the proliferation of the virus. After long-term treatment, the virus may mutate and become resistant. Long-term medication is required, as short-term application has only temporary effects. If it is used in combination with other drugs with different targets, it can improve the efficacy and reduce the occurrence of drug resistance. Other treatment methods such as ribavirin and gene recombinant interference α-2b (intron A) combined, interferon complex, interferon complex (pegasys, 2001) in the treatment of chronic hepatitis C. In addition, in order to control the spread of hepatitis B, the medical community has also developed an effective hepatitis B vaccine and immunized the public. Such as the drug used in the treatment of chronic hepatitis B is α-interferon (IFN), its role is mainly immune regulation. However, interferon treatment of chronic hepatitis B has its constraints: First, it has never been truly successful unless the patient has a substantial immune response to HBV prior to treatment. Second, a sudden, transient exacerbation of hepatitis activity (manifested by elevated ALT) prior to interferon-induced HBeAg seroconversion can lead to liver failure and occasionally death in patients with cirrhosis. A new generation of anti-hepatitis and anti-cirrhosis drugs will be highly competitive in the market. Anti-hepatitis drugs have a very large market at home and abroad, but the current anti-hepatitis drugs generally have the problem of drug resistance. Successfully developed drugs that can solve the drug resistance of anti-hepatitis drugs, have high clinical efficacy, and have low toxic and side effects should have very good market prospects.

So far, there is no report in the prior art that contains formula (I) compound alisol A (Alisol A) as an active ingredient in the treatment of AIDS, and there is no application of this compound in the preparation of hepatitis B medicine and hepatitis B inhibitor medicine. application report.

Summary of the invention: the object of the present invention is to provide a pharmaceutical composition for the treatment of hepatitis B, which contains formula (I) compound alisol A (Alisol A) and a pharmaceutically acceptable carrier and/or excipient in an effective amount for the treatment of hepatitis B The invention provides the application of the pharmaceutical composition in the preparation of anti-hepatitis B medicine and anti-hepatitis B inhibitor medicine.

In order to realize the above-mentioned purpose of the present invention, the present invention provides following technical scheme:

The pharmaceutical composition for treating hepatitis B, which contains the compound of formula (I) Alisol A (Alisol A) as an active ingredient, and contains conventional pharmaceutical carriers.

The present invention also provides the application of the above pharmaceutical composition in the preparation of anti-hepatitis B medicine and anti-hepatitis B inhibitor medicine.

When the compound of the present invention is used as a medicine, it can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90% of the compound of the present invention alisol A (AlisolA), and the rest are pharmaceutically acceptable, non-toxic and inert pharmaceutically acceptable carriers for humans and animals .

The pharmaceutical carrier is one or more selected from solid, semi-solid and liquid diluents, fillers and pharmaceutical product adjuvants. The pharmaceutical composition of the present invention is used in a dose per body weight. The medicine of the present invention can be administered in two forms of injection (intravenous injection, intramuscular injection) and oral administration.

Detailed ways:

The following examples can illustrate the present invention in more detail, but do not limit the present invention in any form.

Example 1: Extraction, separation and structural identification of alisol A

Extraction and separation of alisol A (Alisol A):

Gather Alisma orientalis tuber, whose scientific name is identified as Alisma orientalis (Sam.) Juzep., dry in the shade at room temperature, and pulverize. Take 20 kg of dry powder, reflux extraction with 90% ethanol for 3 times, each time for 3 hours, combine the ethanol extracts, recover under reduced pressure to obtain ethanol extract. The extract was suspended in water, extracted with chloroform and n-butanol respectively, and the solvent was recovered to obtain a chloroform fraction (450 g) and a n-butanol fraction (130 g). The chloroform part was dissolved in methanol and adsorbed on silica gel, placed at room temperature and evaporated to dryness, ground into powder, subjected to silica gel column chromatography, eluted with chloroform-methanol (100:0-90:10) gradient, TLC F254 silica gel thin plate guided separation and detection of each Fractions, the same fractions were combined, the 14th-18th fractions were combined, and methanol recrystallized twice to obtain 23 grams of small white needle crystal compound Alisol A (0.115%). The TLC silica gel F254 thin plate detection is the same as the reference substance Alisol A (Alisol A), and the Rf value is the same.

Structural data of Alisol A:

The melting point was measured with XRC-1 microscopic melting point tester of Keyi Factory of Sichuan University; the infrared spectrum (IR) was measured with Bio-Rad FTS-135 spectrophotometer, and KBr was pressed into tablets. Mass Spectrum (MS) was determined by Auto SPEC3000 mass spectrometer, using EI-MS technology. The nuclear magnetic resonance spectrum ( ¹ H NMR and ¹³ C NMR) was measured with a Bruker WH-400 superconducting nuclear magnetic resonance instrument, TMS was used as an internal standard, and CDCl ₃ was used as a solvent. The 200-300 mesh silica gel produced by Qinghai Chemical Factory was used as the column chromatography material; the silica gel plate produced by Qinghai Chemical Factory was used for thin layer chromatography to guide the separation. Developing solvent: A: chloroform-methanol (95:5, /v): B: petroleum ether-ethyl acetate-(7:3): C, methanol-water (8:2). Color developer: A, 5% H ₂ SO ₄ -EtOH.

Structure: such as chemical formula

shown

Molecular formula: C ₃₀ H ₅₀ O ₅

Molecular weight: 490

Appearance: Colorless columnar crystal (methanol

[α] _D 25: +99° (c 0.586, pyridine).

IR v _max KBr cm ^-1 : 3391 (OH), 1733, 1717, 1652, 1608, 1575, 1506, 1500, 1361, 1248, 1183.

EI-MS m/z:: 472[M] ⁺ .

¹ H NMR (C ₅ D ₅ N): ¹ H-NMR (400MHz, CDCl ₃ ): δ1.02 (3H, d, J=6.9Hz, H-21), 0.99, 1.05, 1.05, 1.07, 1.12, 1.24, 1.30 (each 3H, s), 1.76 (1H, d, J=10.8Hz, H-9), 2.79 (1H, m, H ₁ -12), 3.00 (1H, br.s, H-24) , 3.78 (1H, dd, J=9.4, 2.4Hz, H-23), 3.88 (1H, ddd, J=10.7, 10.7, 6.0Hz, H-11).

¹³ C NMR (CDCl ₃ ) δ: 30.9 (C-1), 33.6 (C-2), 221.0 (C-3), 46.9 (C-4), 48.4 (C-5), 20.1 (C-6) , 34.2(C-7), 40.3(C-8), 49.4(C-9), 36.8(C-10), 69.6(C-11), 34.2(C-12), 137.6(C-13), 56.9(C-14), 30.4(C-15), 29.0(C-16), 135.2(C-17), 22.9(C-18), 25.5(C-19), 28.1(C-20), 20.1 (C-21), 40.0 (C-22), 69.4 (C-23), 77.7 (C-24), 74.1 (C-25), 27.2 (C-26), 26.2 (C-27), 29.5 ( C-28), 19.9(C-29), 24.0(C-30).

Example 2:

The toxicity of Alisol A (Alisol A) to 2.2.15 cells and the inhibitory effect on the secretion of HBsAg and HBeAg.

1 Materials and methods

1.1 Drugs: Alisol A (Alisol A) is a monomer compound from the traditional Chinese medicine Alisma plantago-aquqticavar.orienta.

1.2 2.2.15 Cells were sourced from Beijing 302 Hospital; cultured in DMEM, supplemented with 10% fetal bovine serum, 0.03% L-glutamine, 100mg/LG418, ^{10 5} IU/L penicillin, 100mg/L chain Mycin, 5% NaCO ₃ to adjust the pH to 6.8.

1.3 Instrument: microplate reader ELX800 (Germany)

1.4 Drug application Use trypsin to disperse the 2.2.15 cells into a single cell suspension, divide each well into a 96-well plate at 3×10 ⁴ ·0.1ml ^-1 , and replace with the drug-containing culture medium after 24 hours. For eight drug concentrations, two wells were set up for each concentration, and a control without drug was set up; after 11 days, the supernatant was collected and prepared for the determination of HBsAg and HBeAg. Simultaneous determination of drug cytotoxicity.

1.5 Cytotoxicity detection Drug cytotoxicity was detected according to the MTT method established by Mosmann ^[1] . The specific method is: add 0.1ml/well of 0.4g/L MTT serum-free culture solution to the cell wells where the supernatant was removed, incubate at 37°C for 4h, remove the supernatant, dissolve with dimethyl sulfoxide, and measure at 490nm wavelength The absorbance A value of the absorbance. Destruction percentage η _destroy =(A _{cell group} -A _{experimental group} )/(A _{cell group} -A _{blank group} )×100, 50% toxic concentration (CC50) is the drug concentration when the surviving cells in the experimental wells account for 50% of the control wells.

1.6 Detection of HBsAg and HBeAg: detection by enzyme-linked detection kit produced by Huamei Bioengineering Company. The inhibitory percentage of drug to antigen η _inhibitory =(A _{cell group} -A _{experimental group} )/(A _{cell group} -A _{blank group} )×100, 50% inhibitory concentration (IC50) is HBsAg or HBeAg when the inhibitory rate is 50% drug concentration. Selection index (SI) is a parameter to evaluate the clinical application prospect of the drug, TI=CC50/IC50.

2. Results: Effects of drugs on HBsAg, HBeAg secretion and cell survival. In this experiment, HBsAg and HBeAg were selected as the research indicators for the effect of Alisol A (Alisol A), and the cytotoxicity was detected by MTT method (Table 1), and the therapeutic index was calculated accordingly to evaluate the clinical application prospect of the drug, in which SI>2 was effective Low toxicity, 1<SI<2 means low efficacy and toxicity, TI<1 means toxic effect. Table 1, Table 2 and Table 3 give the specific results.

Table 1 Toxic effect of Alisol A on 2.2.15 cells Compound name concentration OD±S Destruction rate (%) CC ₅₀ Alisol A 1000μg/ml 0.66±0.16 46.77 >1000μg/ml 100 1.16±0.10 6.45 50 1.26±0.14 -1.61 25 1.24±0.10 0.00 12.5 1.15±0.09 7.26 6.25 1.17±0.07 5.65 3.12 1.07±0.04 13.71 1.56 1.27±0.05 -2.42 cell control 1.24±0.07

Table 2. Inhibition of Alisol A (Alisol A) on the secretion of HBsAg and HBeAg in 2.2.15 cells Compound name concentration HBsAg HBeAg OD±SD Inhibition rate(%) OD±SD Inhibition rate(%) Alisitol A 1000μg/ml100502512.56.253.121.56 cell control 0.039±0.010.032±0.010.057±0.010.075±0.020.13±0.010.10±0.120.11±0.030.12±0.010.30±0.08 87.0089.3381.0075.0056.6766.6763.3360.00 0.76±0.020.97±0.090.79±0.120.87±0.1601.17±0.230.80±0.090.73±0.081.01±0.202.05±0.20 62.9352.6861.4657.5642.9360.9864.3950.73

Table 3. The inhibitory effect of alimasol A on HBsAg and HBeAg compound TC50 IC50(HBsAg) IC50(HBeAg) Ti HBsAg HBeAg Alisitol A >1000μg/mL ＜1.56μg/mL 17.47μg/mL 641 >57.24

The results showed that Alisol A could inhibit the secretion of HBsAg and HBeAg from 2.2.15 cells.

3. Conclusion:

The experimental results show that Alisol A (Alisol A) has a certain inhibitory effect on the secretion of HBsAg and HBeAg by 2.2.15 cells in vitro under the half toxicity to 2.2.15 cells.

Example 3:

Inhibitory Effect of Alisol A on Infection of Duck Hepatitis B Virus (DHBV)

Experimental purpose: To study the inhibitory effect of alisol A on duck hepatitis B virus (DHBV) infection.

Experimental Materials:

1. Drugs:

1. Alisol A (Alisol A) is a monomer compound from the traditional Chinese medicine Alisma (Alisma plantago-aquqtica var.orienta). The content in plants is 0.1%.

2. Acyclovir (ACV): produced by Livzon Group Hubei Keyi Pharmaceutical Co., Ltd., batch number 0112005.

2. Animals: male one-day-old Longyan shelducks were purchased from the hatchery of Chaoyang Village, Shijing, and the body weight during the experiment was about 120 grams per duck. They were reared conventionally. Breeding environment: The animal breeding room of the school, the temperature is 24°C-26°C, the relative humidity is 60-80%, regular ventilation, and about 12 hours of light per day. Feed: Baoding 501 duckling feed, produced by the feed factory of Suiping Enterprise Co., Ltd., this product meets the feed hygiene standards. 40kg/bag.

3. Main reagents and instruments:

1. NC membrane: purchased from Amersham Company.

2. DHBV plasmid: extracted by technicians from the virus room of Guangzhou University of Traditional Chinese Medicine Institute of Tropical Diseases.

3. Gap translation kit: purchased from Promega. α- ^32p -dCTP was purchased from Beijing Yahui Company.

4. Sephadex G-50: purchased from Pharmacia.

5. 96-hole hybridization spotter: product of Bio-rad Company in the United States.

6. Geiger counter: product of S.E.International Company in the United States.

4. Positive drug selection:

Acyclovir (ACV), which has a strong inhibitory effect on DHBV, was selected as the positive control drug.

Five, drug dosage preparation:

Alisol A high dose: take 120mg+0.4ml DMSO+3.8ml water (30mg/ml);

Alisol A (Alisol A) low dose; take 80mg+0.5ml DMSO+8.2 water (9.2mg/ml), freshly prepared every day; acyclovir (ACV): 200mg/kg.

6. Results processing: Statistical processing was performed with one-way analysis of variance.

7. Experimental methods and results:

(1), anti-duck hepatitis B virus effect in vivo experiment:

After the one-day-old Longyan shelduck was bought back, blood was collected from the tibial vein on the 6th day. After the serum was separated, the DHBV was detected by dot hybridization, and the congenitally infected ducks were selected for the experiment. Alimasol A (alimasol A) high-dose group and low-dose group were given 1ml/dose/day; another virus control (DHBV) was set up, and water was used instead of drugs; positive drug Acyclovir (ACV) 200mg /kg/day. All the above drugs were administered orally, and a course of treatment was given every 10 days. On day 17 (T ₀ ), day 5 (T ₅ ), day 10 (T ₁₀ ) and day 3 after drug withdrawal (P ₃ ), blood was collected from duck tibial vein, and serum was separated. Freeze at -70°C until testing.

(2), DHBV-DNA detection method:

Take the above-mentioned duck serum, dot the membrane at the same time for each batch, measure the change of the DHBV-DNA level in the duck serum, according to the method in the instruction manual of the gap translation kit, use ³² p to label the DHBV-DNA probe, and do duck serum dot hybridization, autoradiography Membrane spot, measure OD value (filter wavelength is 490nm) on the enzyme label detector, calculate serum DHBV-DNA density.

(3) Experimental results:

Table 4 Inhibitory effect of Alisol A on DHBV-DNA in ducks (x±s) group number of ducks OD ₄₉₀ value T ₀ T ₅ T ₁₀ P ₃ Virus control ACV (200mg/kg/day) low dose (100mg/kg/day) high dose (300mg/kg/day) 6556 1.60±0.361.46±0.671.66±0.570.95±0.18 1.65±0.520.26±0.02 ^* _△△ 1.45±0.700.74±0.34 _△ 1.40±0.560.28±0.08 ^* _△ 1.36±0.620.45±0.11 ^* _△ 1.92±0.592.21±0.001.80±0.390.89±0.55 _△

Note: Alisol A (Alisol A) group was administered at different times compared with before administration (T ₀ ): ^* P＜0.05

Comparison between the Alisol A group and the control group at the same time: _△△ P<0.01 _△ P<0.05

(4), conclusion:

Experiments have shown that two doses of Alisol A (Alisol A) have a 10-day course of treatment, and the high dose (300mg/kg/day) group has a significant inhibitory effect on duck hepatitis B virus.

Example 4:

Alisol A (Alisol A) is first prepared by the method of Example 1, and water for injection is added as usual, finely filtered, potted and sterilized to make an injection.

Example 5:

According to the method of Example 1, Alisol A (Alisol A) was first prepared, dissolved in sterile water for injection, stirred to dissolve, filtered with a sterile suction filter funnel, then sterile fine filtered, and packed in 2 ampoules In the process, after low-temperature freeze-drying, the powder injection was aseptically melt-sealed.

Embodiment 6:

Add the separated Alisol A (Alisol A) to the excipient at a weight ratio of 9:1 to the excipient to prepare a powder.

Embodiment 7:

According to the method of Example 1, Alisol A (Alisol A) was first prepared, and the weight ratio of Alisol A to the excipient was added to the excipient in a ratio of 5:1, and then granulated and compressed into tablets.

Embodiment 8:

Prepare alisol A (Alisol A) earlier by the method for embodiment 1, make oral liquid by conventional oral liquid preparation method.

Embodiment 9:

Alisol A (Alisol A) was first prepared by the method of Example 1, and the weight ratio of Alisol A to the excipient was added to the excipient in a ratio of 5:1 to form a capsule.

Example 10:

Alisol A (Alisol A) was first prepared according to the method of Example 1, and the weight ratio of Alisol A to the excipient was added to the excipient in a ratio of 3:1 to form a capsule.

Claims (3)

1. A pharmaceutical composition for treating hepatitis B, which contains the compound of formula (I) Alisol A (Alisol A) as an active ingredient, and contains a conventional pharmaceutical carrier. 2. The application of the pharmaceutical compound Alisol A (Alisol A) described in claim 1 in the preparation of anti-hepatitis B drugs. 3. The application of the pharmaceutical compound Alisol A (Alisol A) described in claim 1 in the preparation of anti-hepatitis B inhibitor drugs.