CN106310269B

CN106310269B - A pharmaceutical composition with anti-hepatitis C virus effect

Info

Publication number: CN106310269B
Application number: CN201510390220.4A
Authority: CN
Inventors: 孙宏斌; 甄乐; 张帆; 温小安
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2015-07-03
Filing date: 2015-07-03
Publication date: 2021-01-29
Anticipated expiration: 2035-07-03
Also published as: CN106310269A

Abstract

The invention discloses a pharmaceutical composition with an anti-hepatitis C virus effect. The composition comprises nucleoside NS5B polymerase inhibitor I, an NS3/4A serine protease inhibitor and/or an NS5A inhibitor and a pharmaceutically acceptable carrier. The composition has a strong inhibition effect on wild type HCV and various mutant HCV, and the components show obvious synergistic effect, so that a safe and effective novel treatment scheme is provided for preventing and treating chronic hepatitis C, and the composition has a unique development advantage and a wide application prospect.

Description

A pharmaceutical composition with anti-hepatitis C virus effect

Technical Field

The invention belongs to the technical field of medicines, and relates to a novel pharmaceutical composition with an anti-hepatitis C virus effect; in particular, the pharmaceutical composition comprises a hepatitis c virus NS5B polymerase inhibitor and an NS3/4A serine protease inhibitor and/or an NS5A inhibitor.

Background

Hepatitis c is a chronic liver disease caused by Hepatitis C Virus (HCV). After infecting the liver cells, HCV evades host immune surveillance and maintains self-replication by utilizing a lipid metabolic process to initiate chronic infection; and the disorder of the energy metabolism of liver cell substances is caused, and the liver cancer finally progresses to fatty liver, liver cirrhosis and even primary liver cancer. The incidence rate of chronic hepatitis C is high, the degree of chronicity is high, the harmfulness is high, and the chronic hepatitis C becomes a global public health problem; in China, as many as 2500 million HCV-infected patients have the dilemma of low cognition rate, low diagnosis rate and low treatment rate aiming at the high-risk infection.

It is worth noting that patients with hepatitis c can be cured if they can receive the standard antiviral therapy in time. Alternative antiviral treatment regimens with ribavirin in combination with long-acting interferon are still poor candidates for small fractions of infected patients, and are associated with poor compliance, resulting in local, systemic or even psychiatric side effects. In addition, more than half of the patients are not completely cured after the combination therapy and are required to incur high treatment costs. In contrast, since the NS3/4A protease inhibitors Telaprevir and Boceprevir were marketed, the study of antiviral therapies that inhibit key proteins in the HCV life cycle has been a focus in the field. Many anti-HCV Drugs (DAAs) that act directly on key proteins, such as NS3/4A serine protease inhibitors, NS5A inhibitors, NS5B polymerase inhibitors, have been successfully marketed and achieve a complete oral cure for HCV without interferon. However, the existing drug treatment means still have the defects of long treatment period, large drug dosage and high drug price. Therefore, there is a high necessity to develop new anti-HCV drugs to meet clinical needs.

HCV has at least six gene subtypes, each of which is sensitive to drugs to varying degrees; HCV is highly mutated, and further, treatment differences and even drug resistance of the same subtype still exist among different individuals, so that the search for HCV inhibitors which are effective on the whole genotype and have high drug resistance barriers is significant. Currently, the NS3/4A serine protease inhibitors that are used clinically or in clinical trials are mainly Telaprevir, Boceprevir, Danoprevir, Simeprevir, Asunaprevir, Paritaprevir and Grazoprevir; NS5A inhibitors mainly include Daclatatsvir, Ledipasvir, Ombitasvir and Elbasvir; both of these inhibitors are very likely to induce the appearance of drug-resistant HCV in clinical application, resulting in greatly reduced efficacy. In Chinese patent application 201110174871.1, 2 ' -deoxy-2 ' -fluoro-2 ' -methyl nucleoside derivatives, their preparation and use in pharmacy, a series of nucleoside NS5B polymerase inhibitors with anti-HCV effect are disclosed. Although the compound related in the patent can generate stronger inhibitory activity to drug-resistant HCV compared with the existing similar drug Sofosbuvir, the single application of the drug still has the defects of low antiviral activity, large potential virus drug-resistant risk and the like, and cannot meet the treatment requirement.

The best strategy for solving the above problems is to perform the combination administration of inhibitors aiming at different action targets. The drug combination in the form of the pharmaceutical composition can simplify the administration mode, improve the administration compliance, overcome the defects of single administration of each component and further improve the drug effect. Currently, only Harvoni by Gilead and Viekira Pak by AbbVie are FDA approved pharmaceutical compositions on the market. Wherein Harvoni is a fixed dose combination of the nucleoside NS5B polymerase inhibitor Sofosbuvir and the NS5A inhibitor Ledipasvir, and Viekira Pak is a fixed dose combination of the NS3/4A serine protease inhibitor Paritaprevir and the NS5A inhibitor Ombitasvir, but requires the addition of the non-nucleoside NS5B polymerase inhibitor Dasabvir. However, the pharmaceutical composition consisting of nucleoside NS5B polymerase inhibitor and NS3/4A serine protease inhibitor and the pharmaceutical composition consisting of NS5B polymerase inhibitor, NS3/4A serine protease inhibitor and NS5A inhibitor have not been successfully marketed at present.

Disclosure of Invention

In order to overcome the defects of the existing medicines, the invention provides a novel medicine composition which can effectively play a role in resisting HCV, reduce the administration dosage of the existing medicines, shorten the treatment period and reduce the virus drug resistance risk so as to obtain better clinical curative effect.

To achieve the object of the present invention, the present invention provides a novel administration regimen for treating chronic hepatitis C with greater safety and efficacy. The technical scheme relates to a pharmaceutical composition, which comprises a nucleoside NS5B polymerase inhibitor, an NS3/4A serine protease inhibitor and/or an NS5A inhibitor and a pharmaceutically acceptable carrier.

In the above pharmaceutical composition, the nucleoside NS5B polymerase inhibitor is (S) -2- (((S) - (((2R, 3R, 4R, 5R) -5- (4-n-butylamido-2-oxopyrimidin-1 (2H) -yl) -4-fluoro-3-hydroxy-4-methyl-tetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) amino) isopropyl propionate I-1, (S) -2- (((S) - ((2R, 3R, 4R, 5R) -5- (4-n-valerylamino-2-oxopyrimidin-1 (2H) -yl) -4-fluoro-3-hydroxy-4-methyl-tetrahydrofuran-2-yl) methoxy) (phenoxy) Phenyl) phosphoryl) amino) isopropyl propionate I-2, (S) -2- (((S) - (((2R, 3R, 4R, 5R) -5- (4-n-hexanoylamido-2-oxopyrimidin-1 (2H) -yl) -4-fluoro-3-hydroxy-4-methyl-tetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) amino) isopropyl propionate I-3 or (S) -2- (((S) - ((2R, 3R, 4R, 5R) -5- (4-n-hexanoylamido-2-oxopyrimidin-1 (2H) -yl) -4-fluoro-3-hydroxy-4-methyl-tetrahydrofuran-2-yl) methoxy) (phenoxy) phosphorus Acyl) amino) isopropyl propionate I-4, whose structure is shown below;

wherein the preferred NS5B polymerase inhibitor is I-1.

In the above pharmaceutical composition, the NS3/4A serine protease inhibitor is Telaprevir, Boceprevir, Danoprevir, Simeprevir, Asunaprevir, Paritaprevir or Grazoprevir, and has the following structure:

among them, the NS3/4A serine protease inhibitor is preferably Simeprevir.

In the above pharmaceutical composition, the NS5A inhibitor is Daclatasvir, Ledipasvir, Ombitasvir or Elbasvir, and the structure thereof is as follows:

among them, the NS5A inhibitor is Daclatasvir.

In the pharmaceutical composition, the concentration ratio of the NS5B inhibitor I to the NS3/4A inhibitor is 1: 1/5000-100; preferably 1 to (1/2000-10); more preferably 1: (1/600-1).

In the pharmaceutical composition, the concentration ratio of the NS5B inhibitor I to the NS5A inhibitor is 1: 1/50000-10; preferably 1 to (1/20000-1); more preferably 1: (1/6000-0.1).

In the pharmaceutical composition, the concentration ratio of the NS5B inhibitor I to the NS3/4A inhibitor to the NS5A inhibitor is 1 to (1/5000-100) to (1/50000-10); preferably 1 to (1/2000-10) to (1/20000-1); more preferably 1 to (1/600-1) to (1/6000-0.1).

In the pharmaceutical composition, the concentration ratio of the NS5B inhibitor I-1 to the Simeprevir is preferably 1 to (1/5000-100); preferably 1 to (1/2000-10); more preferably 1: (1/600-1).

In the pharmaceutical composition, the concentration ratio of the NS5B inhibitor I-1 to the Daclatatasvir is preferably 1: 1/50000-10; preferably 1 to (1/20000-1); more preferably 1: (1/6000-0.1).

In the pharmaceutical composition, the concentration ratio of the NS5B inhibitor I-1 to the Simeprevir and the Daclatatasvir is preferably 1 to (1/5000-100) to (1/50000-10); preferably 1 to (1/2000-10) to (1/20000-1); more preferably 1 to (1/600-1) to (1/6000-0.1).

The invention also provides application of the pharmaceutical composition in preparing anti-hepatitis C drugs. Converting the concentration ratio into mass ratio, mixing the above components, directly or indirectly adding pharmaceutically acceptable carrier, and making into tablet, capsule, granule, powder, syrup, oral liquid or injection by known operation method.

Surprisingly, the pharmaceutical composition has obvious advantages, which are mainly reflected in the following aspects:

the invention provides a novel drug composition administration scheme containing nucleoside NS5B inhibitor I for the first time.

The technical scheme of the invention realizes unpredictable antiviral effects: at the cellular level of the HCV replicon, it was shown that the nucleoside NS5B inhibitor I, in combination with either NS3/4A inhibitor or NS5A inhibitor, showed unexpected synergy; in the existing pharmaceutical compositions, no obvious synergistic effect exists.

The NS5B inhibitor I, the NS3/4A inhibitor and the NS5A inhibitor are combined for use, and compared with the single administration or the combined administration of any two of the components, the antiviral effect can be obviously improved, and the dosage is greatly reduced.

The activity of mutant HCV replicon cells shows that the pharmaceutical composition provided by the invention can generate effective inhibition effect on mutant HCV and maintain higher antiviral level. This suggests that the present invention can effectively prevent the occurrence of various drug-resistant variants of HCV in clinical applications.

Drawings

FIG. 1 testing of drug resistance Properties of pharmaceutical compositions

Detailed Description

For a better understanding and verification of the reliability of the invention, it is explained in detail below with reference to specific examples, without restricting the invention in any way.

Example 1 combination of NS5B inhibitor I with NS3/4A inhibitor or NS5A inhibitor

Medicine preparation: NS5B inhibitors I-1, Simeprevir, Daclatasvir.

Cell model: 1b genotype HCV subgenomic replicon cell system (Conl cells).

The experimental steps are as follows:

1. co-incubation: replicon cells were seeded into 96-well plates, 8000 cells per well; in compound I-1 of different concentrations (0, 25, 50, 100, 200, 400, 800, 1600nM), different concentrations of Simeprevir (0, 3, 6, 12, 24nM) or Daclatatsvir (0, 0.02, 0.04, 0.08, 0.16nM) were added, and each group of compounds was tested in duplicate wells with a final DMSO concentration of 0.5%; the culture was carried out in a carbon dioxide incubator for 72 hours (see Table 1 for the concentrations of the components of the pharmaceutical composition).

2. And adding a cell viability detection reagent into each hole of cells, and detecting the cell viability. Luciferase substrate Bright-Glo was then added to the cells and after 5 minutes luciferase expression levels were detected using a chemiluminescent detection system to obtain the percentage of replicon inhibitory activity.

3. The Combination Index (CI) was calculated using the Compusyn software, and the combined effect of the pharmaceutical compositions was evaluated by CI value.

Results of the experiment

1. None of the compounds showed cytotoxicity.

2-antiviral activity and synergy evaluation are shown in tables 1-4:

TABLE 1 inhibitory Activity of combinations of I-1 and Simeprevir on HCV I-1(nM) at different concentrations

TABLE 2 CI values for different concentration ratios in combination with Simeprevir for I-1

TABLE 3 inhibitory Activity of combinations of I-1 and Daclatatasvir against HCV Compound I-1(nM) at different concentrations

TABLE 4 CI values for different concentration ratios in combination of I-1 and Daclatasvir

The data reported in the tables are the inhibition rates of the pharmaceutical compositions against HCV subgenomic replicons at different concentrations. And (3) processing the experimental data by utilizing Compuyn software to obtain the Combination Index (CI) of the I-1 and Simeprevir or Daclatatasvir under different concentration ratios. When CI is more than 1, antagonism exists in the medicine components; when CI ═ 1, the two drug components exhibit additive effects; and when CI is less than 1, the medicinal components have synergistic effect. As is apparent from tables 2 and 4, I-1 and Simeprevir show synergistic effects of different degrees in different concentration ratios; but has stronger synergistic effect with Daclatatasvir.

The experimental results fully show that in the novel pharmaceutical composition provided by the invention, the NS5B polymerase inhibitor and the NS3/4A serine protease inhibitor or the NS5A inhibitor can play a synergistic effect; the composition shows strong antiviral effect when used in combination; this experiment confirms the rationality of the technical scheme of the present invention and hopefully reduces the administration dosage of the existing drugs in clinical application and enhances the anti-HCV activity to obtain better therapeutic effect.

Example 2 combination of NS5B inhibitor I with NS3/4A inhibitor and NS5A inhibitor

Medicine preparation: NS5B inhibitors I-1, Simeprevir, Daclatasvir.

Cell model: 1b genotype HCV subgenomic replicon cell system (Conl cells).

The experimental steps are as follows:

1. preparation of a pharmaceutical composition solution: NS5B inhibitor I-1, Simeprevir and Daclatatasvir are mixed at the concentration ratio of 1: 0.015: 0.000075 to prepare a pharmaceutical composition solution S1; NS5B inhibitor I-1, Simeprevir and Daclatatasvir are mixed at the concentration ratio of 1: 0.015: 0.00015, and a pharmaceutical composition solution S2 is prepared; NS5B inhibitor I-1, Simeprevir and Daclatatasvir are mixed at the concentration ratio of 1: 0.0075: 0.000075 to prepare a medicinal composition solution S3; preparing a pharmaceutical composition solution S4 with the concentration ratio of Simeprevir to Daclatatasvir being 200: 1; wherein the initial concentration of NS5B inhibitor I-1 in S1-3 was 10. mu.M, and the initial concentration of Simeprevir in S4 was 10. mu.M.

2. Co-incubation: replicon cells were seeded into 96-well plates, 8000 cells per well; diluting the pharmaceutical composition S1-4 with different concentration ratios according to a 3-fold concentration gradient, incubating with cells, and detecting by double-hole detection, wherein the final concentration of DMSO is 0.5%; the carbon dioxide incubator is used for 72 hours.

3. And adding a cell viability detection reagent into each hole of cells, and detecting the cell viability. The luciferase substrate Bright-Glo was then added to the cells and after 5 minutes the luciferase expression level was detected using a chemiluminescent detection system, thus obtaining the inhibition curve of pharmaceutical composition S1-4 against HCV replicon.

Results of the experiment

1. None of the compounds showed cytotoxicity.

2. Antiviral activity is shown in table 5: EC of each of I-1, Simeprevir, and Daclatatsvir₅₀、EC₉₀、EC₉₅、EC₉₉As shown in table 6.

TABLE 5 combination of I-1 with Simeprevir and Daclatatsvir

TABLE 6 anti-HCV Activity of I-1, Simeprevir and Daclatatasvir

First, in the inhibition curve of S1, I-1, Simeprevir and Daclatatasvir EC were selected₅₀Concentration ratio Com A nearby and higher than EC₅₀Twice the concentration ratio Com D; in the inhibition curve of S2, the concentration of Daclatatasvir was chosen as its EC₅₀Com B is prepared at twice concentration; in the inhibition curve of S3, the EC of both I-1 and Daclatasvir are selected₅₀Com C is prepared at twice concentration; selecting EC of S4 in inhibition curve when each component drug is used alone₅₀Com E in the near concentration ratio and EC higher than each component drug₅₀Com F is prepared at twice the concentration. The concentration ratios are detailed in Table 5Com A-F, and the anti-HCV effect of the pharmaceutical composition of the present invention is evaluated by comparing the inhibition rates of different concentration ratios to HCV.

The experimental result shows that the inhibition rate of Com A on HCV replicon reaches 82.91%, the inhibition rate of Com E reaches 73.14%, and the inhibition rate of the Com A alone on HCV replicon is only about 50%. This indicates that the anti-HCV effect of the combination is much stronger than that of the single administration at the same concentration of the administration.

Secondly, the inhibition rate of Com B on HCV reaches 91.06 percent, which is compared with EC when alone is administrated₉₀Compared with the prior art, the administration concentration of the components is obviously reduced when the components are used together; similarly, the inhibition rate of Com C on HCV is more than 95%, and the concentration of each component is far less than that of EC₉₅. Both sets of data demonstrate that the pharmaceutical composition can significantly reduce the administered concentration (dose) with the same anti-HCV effect.

When the concentrations of the three components are all increased to EC₅₀After twice, the inhibition rate of Com D is as high as 99.5%, while the inhibition rate of the two-component Com F is only 89.55%, which further proves that the pharmaceutical composition can significantly reduce the administration concentration; in addition, the invention further indicates that the antiviral effect can be improved by adding the nucleoside NS5B inhibitor I into the pharmaceutical composition.

The experiments surprisingly prove that the pharmaceutical composition, namely the NS5B inhibitor I, NS3/4A inhibitor and the NS5A inhibitor combined drug, can remarkably improve the antiviral effect compared with the single administration or the combined administration of any two components, greatly reduces the dosage and has far better effect than the known pharmaceutical composition.

EXAMPLE 3 testing of drug resistance Properties of pharmaceutical compositions

Medicine preparation: NS5B inhibitors I-1, Simeprevir, Daclatasvir.

Cell model: a mutant HCV subgenomic replicon cell system. Including S282TNS5B mutant replicon, R155K NS3/4A mutant replicon, Q80K NS3/4A mutant replicon, and Y93H NS5A mutant replicon.

The experimental steps are as follows:

1. preparation of a pharmaceutical composition solution: the ratio of the pharmaceutical composition solution S1 to the pharmaceutical composition solution S4 was the same as in example 2; the concentration ratio of the NS5B inhibitor I-1 to the Simeprevir is 1: 0.015, and a pharmaceutical composition solution S5 is prepared; the concentration ratio of the NS5B inhibitor I-1 to the Daclatatasvir is 1: 0.000075, and a pharmaceutical composition solution S6 is prepared; wherein the initial concentration of NS5B inhibitor I-1 in S1, 5, 6 was 10. mu.M, and the initial concentration of Simeprevir in S4 was 10. mu.M.

2. Co-incubation: inoculating the wild type and variant replicon cells into a 96-well plate, 8000 cells per well; diluting the drug or the drug composition S1 and S4-6 with three times of concentration, respectively co-incubating the drug or the drug composition with two replicon cells, and carrying out double-hole detection, wherein the final concentration of DMSO is 0.5%; culturing for 72 hours in a carbon dioxide incubator;

3. and adding a cell viability detection reagent into each hole of cells, and detecting the cell viability. Adding luciferase substrate Bright-Glo into the cells, detecting luciferase expression level with chemiluminescence detection system after 5 min, and calculating to obtain half effective concentration EC₅₀And a Fold Change value, wherein the wild-type replicon inhibitory activity is EC_WTMutant replicon inhibitory Activity is EC_Mut，Fold Change＝EC_Mut/EC_WT；

3. Fold Change was used to measure the sensitivity of variant HCV to a drug or pharmaceutical composition. When the Fold Change > 1.2, it is considered that the HCV is less sensitive to the drug and the drug resistance phenomenon occurs.

The experimental results are shown in fig. 1:

as can be seen from FIG. 1, when each component was administered alone, I-1 had a reduced inhibitory activity against the S282T NS5B mutant replicon; the inhibitory activity of Simeprevir to R155KNS3/4A mutant and Q80KNS3/4A mutant replicons is obviously reduced; daclatasvir also showed a significant reduction in inhibitory activity against the Y93H NS5A mutant replicon.

The pharmaceutical composition Com A has ideal inhibitory activity on 4 selected variants of HCV; in contrast, the R155K, Q80K, Y93H variant HCV still developed a certain degree of resistance to composition Com E containing only Simeprevir and Daclatasvir.

The experiments prove that the pharmaceutical composition can generate good inhibition effect on wild type HCV and various mutant HCV; the experiment indicates that the drug composition can effectively prevent the generation of drug-resistant virus.

EXAMPLE 4 tablets

The powders of the above formula were mixed and compressed by a tablet press to make 1200mg tablets per tablet. The tablets may be film coated or sugar coated as desired.

EXAMPLE 5 tablets

EXAMPLE 6 tablets

Claims

1. A pharmaceutical composition comprising NS5B polymerase inhibitor I-1 and an NS3/4A serine protease inhibitor and/or an NS5A inhibitor;

the structure of the NS5B polymerase inhibitor I-1 is shown as follows:

；

the NS3/4A serine protease inhibitor is Simeprevir;

the NS5A inhibitor is Daclatatasvir;

wherein the concentration ratio of the NS5B polymerase inhibitor I-1 to the NS3/4A serine protease inhibitor is 1: (1/600-1); the concentration ratio of the NS5B polymerase inhibitor I-1 to the NS5A inhibitor is 1: 1/6000-0.1; the concentration ratio of NS5B polymerase inhibitor I-1 to NS3/4A serine protease inhibitor and NS5A inhibitor is 1: 0.015: 0.000075 or 1: 0.0075: 0.000075 or 1: 0.015: 0.00015.

2. Use of the pharmaceutical composition of claim 1 for the preparation of an anti-hepatitis c medicament.

3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is a tablet, capsule, granule, powder, syrup, oral liquid, or injection.