Pharmaceutical composition for treating hepatitis, liver fibrosis, and liver cancer

Abstract

The present invention relates to a pharmaceutical composition for treating hepatitis, liver fibrosis, and liver cancer, which comprises the following components in parts by weight: 0.15-10 parts of vinegar-baked bupleurum root polysaccharides or bupleurum root polysaccharides and 0.05-5 parts of a drug for treating hepatitis.

Claims

1. A pharmaceutical composition for treating hepatitis, liver fibrosis, and liver cancer, comprising the following components in parts by weight: 0.15-10 parts of vinegar-baked bupleurum root polysaccharides or bupleurum root polysaccharides; and 0.05-5 parts of matrine or Dipivoxil for treating hepatitis.

2. A pharmaceutical composition for treating hepatitis, liver fibrosis, and liver cancer according to claim 1, comprising the following components in parts by weight: 0.05-2 parts of matrine; and 0.5-5 parts of vinegar-baked bupleurum root polysaccharides or bupleurum root polysaccharides.

3. A pharmaceutical composition for treating hepatitis, liver fibrosis, and liver cancer according to claim 1, comprising the following components in parts by weight: 0.05-1 part of matrine; and 0.5-5 parts of vinegar-baked bupleurum root polysaccharides.

4. A pharmaceutical composition for treating hepatitis, liver fibrosis, and liver cancer according to claim 1, comprising the following components in parts by weight: 0.010-0.1 part of Dipivoxil; and 0.15-5 parts of vinegar-baked bupleurum root polysaccharides.

5. A pharmaceutical composition for treating hepatitis, liver fibrosis, and liver cancer according to claim 1, comprising the following components in parts by weight: 0.05-2 parts of matrine; and 0.84 part of vinegar-baked bupleurum root polysaccharides.

6. A traditional Chinese medicine preparation for treating hepatitis and liver cancer, comprising a therapeutically effective amount of a traditional Chinese medicine composition for treating hepatitis and liver cancer according to claim 1 and a pharmaceutically acceptable carrier.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(1) The present invention is described in further detail below by way of examples. The examples are provided to illustrate, instead of limiting the scope of the present invention.

Example 1

(2) Composition:

(3) 60 parts of bupleurum root, 5 parts of oxymatrine (purity 80%), and 20 parts of starch

(4) Preparation Method

(5) The bupleurum root is weighed and added to water, the weight of the water is 10 times the weight of the bupleurum root, soaked for 30 min, then boiled, and simmered for 30 min, filtered with gauze, and boiled for another 30 min. The filtrates is combined, concentrated, cooled, added with absolute ethanol to give a volume fraction of ethanol of 80%, and then stood at room temperature for 24 h. The suspension is centrifuged for 5 min at 4000 r/min. The pellet is the bupleurum root polysaccharides (PSS). Oxymatrine and starch are added to the pellet, after that the pellet is prepared into tablets through a method well known in the art.

Example 2

(6) Composition

(7) 3 parts of vinegar-baked bupleurum root, 5 parts of oxymatrine (purity 80%), and 10 parts of microcrystalline cellulose;

(8) Preparation Method:

(9) Following the method described in Example 1, vinegar-baked bupleurum root is weighed, extracted with water and precipitated in ethanol. The precipitate was collected by centrifugation, added with oxymatrine and microcrystalline cellulose, and then prepared into capsules through a method well known in the art.

Example 3

(10) Composition

(11) 10 parts of bupleurum root, 1 part of Lamivudine, and 5 parts of lactose. The preparation method is the same as that in Example 1.

Example 4

(12) 15 parts of vinegar-baked bupleurum root, 0.5 part of Adefovir Dipivoxil, and 1 part of lactose. The preparation method is the same as that in Example 1.

Example 5

(13) 50 parts of bupleurum root, 2 parts of Entecavir, and 5 parts of microcrystalline cellulose. The preparation method is the same as that in Example 1.

Example 6

(14) 40 parts of vinegar-baked bupleurum root, 1.5 parts of Telbivudine, and 15 parts of microcrystalline cellulose. The preparation method is the same as that in Example 1.

Example 7

(15) 35 parts of vinegar-baked bupleurum root, 2 parts of Tenofovir, and 10 parts of microcrystalline cellulose. The preparation method is the same as that in Example 1.

Example 8

(16) The pharmaceutical composition of the present invention enhances the distribution of oxymatrine (OM) in the rat liver.

(17) 36 rats are acclimated for 7 days, and randomized into a treatment group with oxymatrine (OM) alone, and a treatment group with a combined drug of the present invention (drug in Example 1).

(18) Wherein, the dosage of oxymatrine (OM) is 54 mg/kg, and the bupleurum root polysaccharides are used at a corresponding dosage in Example 1. After a single administration, blood is sampled from the abdominal aorta respectively at 20 min, 60 min, and 240 min. The rat is sacrificed, from which the liver, spleen, lung, kidney, and heart are removed. The blood is wiped off with filter paper, and the adipose tissue is removed. 0.1 mL of serum is extracted with chloroform and blow dried with nitrogen, and the residue is dissolved in 0.1 mL of methanol and used as a test solution. The tissue sample is accurately weighed, added with physiological saline at a ratio of 1:10, and homogenized. 0.2 mL of the homogenate is taken, and treated following method the same as the blood sample. The concentrations of oxymatrine (OM) and its metabolite (M) are determined by LC-MS, the content in the tissue and the area under the curve are calculated. The results are shown in Tables 1 and 2 below.

(19) TABLE-US-00001 TABLE 1 AUC results of oxymatrine (g/L*h, n = 6) Tissue Group Plasma Liver Spleen Lung Kidney Heart OM 520.50 125.02 64.94 365.52 69.66 125.35 OM + PSS 253.96 163.82 34.79 439.25 72.08 111.47

(20) TABLE-US-00002 TABLE 2 AUC results of matrine (g/L*h, n = 6) Tissue Group Plasma Liver Spleen Lung Kidney Heart OM 15253.01 3611.03 6402.53 6448.48 5602.98 1963.81 OM + PSS 8221.87 2808.82 3496.18 2521.83 3506.81 1256.42

(21) As calculated, the rates of targeting to liver of OM, M and the two in combination are 2.37, 1.42, and 1.46 times, respectively. The result indicates that the bupleurum root polysaccharides (PSS) can significantly increase the distribution of orally taken oxymatrine preparations in the liver, thereby exerting a better liver protection effect.

Example 9

(22) The pharmaceutical composition of the present invention enhances the Lamivudine level in the rat liver.

(23) Grouping of the test animals and administration are the same as the Example 1 (except that the drug used is the same as Example 3). The dosage of Lamivudine is 15 mg/kg, and the bupleurum root polysaccharides are at a corresponding dosage.

(24) The Lamivudine content was determined by liquid chromatography. 0.1 mL of plasma is added with 10 L of 0.2 mol ammonium acetate, and then with 1 mL of acetonitrile, vortexed, and centrifuged. The supernatant is collected and injected for determination. 0.1 g of the tissue sample is added with 1 mL of physiological saline, homogenized, and treated following the same method as for the plasma sample. The Lamivudine contents in the plasma and tissue sample are determined. The results are shown below.

(25) TABLE-US-00003 TABLE 3 AUC results of Lamivudine (g/L*h, n = 6) Plas- Liv- Kid- Stom- Group ma er Spleen Lung ney Heart ach Lamivudine 25.36 27.32 30.25 23.11 24.32 26.75 28.78 Lamivudine + 15.23 36.78 16.25 10.21 19.65 18.34 15.32 bupleurum root polysaccharides

(26) As can be seen from Table 3, the bupleurum root polysaccharides can significantly increase the intrahepatic distribution of lamivudine, and are expected to reduce the adverse reactions such as nausea and vomiting.

Example 10

(27) The pharmaceutical composition of the present invention enhances the inhibition of Adefovir Dipivoxil on the replication of HBV.

(28) The drug of Example 4 is used. The concentration of Adefovir Dipivoxil was 1 mol, and the vinegar-baked bupleurum root polysaccharides are at a medium dosage in corresponding dosage, one-half of the medium dosage is a low dosage, and twice of the medium dosage is a high dosage.

(29) The experiment is started with HepG2.215 cells that were grown to 80% confluence.

(30) The cells were assigned to 5 groups, comprising a blank control group, a treatment group with Adefovir Dipivoxil, a treatment group with Adefovir Dipivoxil in combination with high dosage of vinegar-baked bupleurum root polysaccharides, a treatment group with Adefovir Dipivoxil in combination with medium dosage of vinegar-baked bupleurum root polysaccharides, and a treatment group with Adefovir Dipivoxil in combination with low dosage of vinegar-baked bupleurum root polysaccharides. Culturing for 48 h after administration, the supernatant is collected, the expression of the HBV gene is determined by fluorescent quantitative PCR, and the HBsAg and HBeAg concentrations are determined by ELISA. The results are shown in Table 4.

(31) TABLE-US-00004 TABLE 4 Effect of the preparation of the present invention on replication of HBV (normalization method, 100% for control group) Adefovir Adefovir Adefovir Blank Dipivoxil + Dipivoxil + Dipivoxil + control Adefovir low dosage medium dosage high dosage group Dipivoxil of PSS of PSS of PSS HBsAg 100 35 1.2 25 2.5 15 3.6 5 2.7 HBeAg 100 40 3.4 30 3.5 20 3.2 10 2.5 Expression 100 50 5.3 30 3.6 20 2.5 11 2.3 of HBV gene

(32) As can be seen from the table above, the vinegar-baked bupleurum root polysaccharides can significantly enhance the inhibition of adefovir dipivoxil on the replication of HBV, with the inhibition rate being increased from 60-65% to 5-10%, and the inhibition rate for gene expression being increased from 50% to nearly 90%.

Example 11

(33) The drug of the present invention enhances the effect of Telbivudine against duck hepatitis B virus (DHBV).

(34) The drug in Example 6 is used.

(35) One-day-old ducklings are injected with DHBV-DNA-positive duck serum via the tibial veins (0.3 mL each), and blood is taken 7 days after infection. The serum is separated and the serum DHBV-DNA content is detected. The duckling serum is tested to be DHBV positive. Then, the ducks are randomly divided into 5 groups, comprising a virus control group, a treatment group with Telbivudine (80 mg.Math.kg.sup.1), a treatment group with Telbivudine in combination with high dosage of PSS, a treatment group with Telbivudine in combination with medium dosage of PSS, and a treatment group with Telbivudine in combination with low dosage of PSS, each group has 6 ducks. The medium dosage of vinegar-baked bupleurum root polysaccharides is as the dosage in Example 6, the low dosage is one-half of the medium dosage, and the high dosage is twice of the medium dosage. The ducks are administered by gastric perfusion twice a day. The virus control group is given the same volume of normal saline for 10 consecutive days. Blood samples are collected from the tibial veins of the duck before administration (T0), at day 5 (T5) and day 10 (T10) of the administration, and 3 days after withdrawal (P3). The serum is separated and stored at 70 C. until test. The serum of the ducks to be tested is taken and dotted at the same time to determine the dynamics of the DHBV-DNA level in the duck serum. According to the instructions for the kit, a 32P labeled DHBV-DNA probe is used, and dot blotting is performed on the duck serum. The dot is radioactively self-developed. A value (the optical filter is at 490 nm) is determined on a microplate reader, and the absorbance of serum DHBV-DNA is calculated. The A value from dot blotting is used as the DHBV-DNA level of the specimen. The mean values of the serum DHBV-DM level in the same group of ducks before and after administration are compared, and the rates of inhibition on serum DHBV-DNA in each treatment group at various times are calculated. The results are shown in Table 5.

(36) TABLE-US-00005 TABLE 5 Inhibition of the inhibitor of the present invention on the replication of HBV (normalization method, 100% for control group) Blank Telbivudine + Telbivudine + Telbivudine + control low dosage medium dosage high dosage group Telbivudine of PSS of PSS of PSS HBsAg 100 30 1.3 21 2.1 12 2.6 3.6 1.7 HBeAg 100 37.2 3.4 28.6 2.5 20.9 2.3 9.5 + 1.2 Expression of 100 50.5 5.3 26.7 1.3 19.8 2.8 9.6 1.3 HBV gene

(37) As can be seen from Table 5, the preparation of the present invention has a good inhibitory effect on the replication of DHBV, and the result is obviously superior to telbivudine that is used alone.

Example 12

(38) The bupleurum root polysaccharides enhance the distribution of Entecavir in liver, and reduce its distribution in other tissues.

(39) The drug of Example 5 is used.

(40) 20 male mice are fasted for one night, but free accessed to water before the experiment. The animals are divided into three groups according to body weight, including a treatment group with Entecavir, and treatment groups with bupleurum root polysaccharides combined with Entecavir. The animals are administered for 5 consecutive days. The dosage of Entecavir is 0.1 mg/kg, and the corresponding dosage of bupleurum root polysaccharides in Example 5 is a high dosage, one-half of which is a low dosage. The animals are sacrificed after the last administration and the blood, heart, liver, spleen, lung, kidney, intestine, stomach, bone marrow, and brain are taken. 100 L of plasma is directly precipitated by adding 800 L of acetonitrile, and the supernatant is frozen and centrifuged. 500 L of the supernatant is blow dried with nitrogen, and 100 L of the mobile phase is added for reconstitution, and injected for analysis. 0.1 g of the tissue is added with 1 mL of water and homogenized. The homogenate is treated following the treatment method for the plasma. The in vivo analysis results are shown in Table 6.

(41) Effect of bupleurum root polysaccharides on in vivo distribution of Entecavir (ng/mL)

(42) TABLE-US-00006 TABLE 6 Test result of Entecavir concentration (ng/L, n = 6) Group Plasma Liver Spleen Lung Kidney Heart Stomach Instine Marrow Entecavir 30.6 26.8 28.4 26.3 27.5 26.14 25.6 24.3 18.9 Entecavir + 23.3 35.4.5 18.3 24.6 23.6 18.9 19.8 18.2 13.4 high dosage of bupleurum root polysaccharides

(43) It can be seen from the table above that bupleurum root polysaccharides can significantly increase the intra-hepatic distribution of Entecavir and reduce its concentration in other tissues, and expected to achieve an efficacy enhancing and toxicity reducing effect.

Example 13

(44) The drug of the present invention enhances the inhibition of Tenofovir on the replication of HBV.

(45) The drug of Example 7 is used.

(46) The experiment is started with HepG2.215 cells that are grown to 80% confluence. The cells were assigned to 5 groups, comprising a blank control group, a treatment group with Tenofovir, a treatment group with Tenofovir in combination with high, medium, and low dosage of vinegar-baked bupleurum root polysaccharides. The concentration of Tenofovir is 0.8 mol, the medium dosage of vinegar-baked bupleurum root polysaccharides is a corresponding dosage in Example 7, one-half of which is the low dosage, and twice of which is the high dosage. Culturing for 48 h after administration, the supernatant is collected, the expression of the HBV gene is determined by fluorescent quantitative PCR, and the HBsAg and HBeAg concentrations are determined by ELISA. The results are shown in Table 7.

(47) TABLE-US-00007 TABLE 7 Effect of the preparation of the present invention on replication of HBV (normalization method, 100% for control group) Blank Tenofovir + Tenofovir + Tenofovir + control low dosage medium dosage high dosage group Tenofovir of PSS of PSS of PSS HBsAg 100 33.2 1.2 25.6 2.5 14.6 3.6 4.5 2.7 HBeAg 100 39.8 2.4 29.8 2.1 18.7 2.2 9.8 2.3 Expression 100 45.2 5.3 31.3 3.6 19.2 2.2 10.8 2.4 of HBV gene

(48) As can be seen from the table above, the vinegar-baked bupleurum root polysaccharides can enhance the potency of Tenofovir against HBV, and are expected to increase the efficacy.

(49) The above description of the detailed embodiments is only to illustrate the preferred implementation according to the present invention, and it is not to limit the scope of the present invention. Accordingly, all modifications and variations completed by those with ordinary skill in the art should fall within the scope of the present invention defined by the appended claims.