WO2023142318A1

WO2023142318A1 - Pharmaceutical composition for treating viral hepatitis

Info

Publication number: WO2023142318A1
Application number: PCT/CN2022/094175
Authority: WO
Inventors: 李瑛颖; 陈明键; 仇思念
Original assignee: 中以海德人工智能药物研发股份有限公司
Priority date: 2022-01-27
Filing date: 2022-05-20
Publication date: 2023-08-03
Also published as: CN114159572A; CN114159572B

Abstract

Disclosed is a pharmaceutical composition for treating viral hepatitis, the pharmaceutical composition comprising a non-steroidal anti-inflammatory drug and an mTOR inhibitor. The pharmaceutical composition is used for treating or preventing viral hepatitis, especially hepatitis B.

Description

A kind of pharmaceutical composition for treating viral hepatitis

technical field

The present disclosure relates to the technical field of antiviral drugs, in particular, to a compound for treating or preventing viral hepatitis, a pharmaceutical composition and applications thereof.

Background technique

Human hepatitis B virus (HBV) infection is an important public health problem worldwide. After acute hepatitis B virus infection, about 8% still develop into chronic hepatitis B infection, and persistent HBV infection will lead to liver cirrhosis and even liver cancer. Hepatitis B transmission is mainly through vertical transmission and horizontal transmission. Vertical transmission refers to mother-to-child transmission; horizontal transmission is mainly through blood.

The treatment of hepatitis B is also a long-term process. The goal of treatment is to suppress or eliminate HBV to the greatest extent, reduce inflammation and necrosis of liver cells and liver fibrosis, delay and prevent disease progression, reduce and prevent liver decompensation, liver cirrhosis, occurrence of HCC and its complications, thereby improving quality of life and prolonging survival time.

Currently, there are many hepatitis B therapeutic drugs on the market, mainly through the use of interferon or nucleoside analogues for antiviral treatment. For interferon, recombinant DNA leukocyte interferon (IFN-α) can inhibit the replication of HBV. However, when interferon is used to treat hepatitis B, it is often accompanied by strong adverse reactions, including bone marrow suppression, affecting thyroid function and depression.

Nucleoside analogs mainly inhibit HBV production by inhibiting the reverse transcriptase activity during HBV replication. Clinically available drugs include the following categories: lamivudine, famciclovir, such as acyclovir, adefovir, entecavir, tenox Fovir, foscarnet sodium, etc., these drugs have a certain inhibitory effect on HBV.

Although these reverse transcriptase inhibitors can effectively reduce the level of HBV DNA and enable patients to control the level of hepatitis B virus, they have no direct effect on the clearance of HBeAg and HBsAg because their target is the process of reverse transcription of RNA into DNA. Therefore, the probability of HBeAg and HBsAg seroconversion in nucleoside analog monotherapy is extremely low, and it cannot really cure hepatitis B. Patients need to take drugs for a long time or even life.

Although the above-mentioned reverse transcriptase inhibitors can enable patients to control the level of hepatitis B virus, problems such as drug resistance, huge medical expenses, and serious side effects of drugs cannot be underestimated. The point is that there is still no drug that can completely clear the virus and achieve a functional cure for hepatitis B. Therefore, there is an urgent need in this field to provide a new drug for treating hepatitis B that does not require long-term or even life-long administration and achieves functional cure.

NSAIDs (Nonsteroidal Antiinflammatory Drugs, NSAIDs) are a class of anti-inflammatory drugs that do not contain a steroidal structure. Since aspirin was first synthesized in 1898, more than 100 kinds of NSAIDs have been listed on the market for more than 100 years. Such drugs include aspirin, acetaminophen, indomethacin, naproxen, naproxen, diclofenac, ibuprofen, nimesulide, rofecoxib, celecoxib, etc. Anti-inflammatory, anti-rheumatic, analgesic, antipyretic and anti-coagulant effects are widely used clinically to relieve osteoarthritis, rheumatoid arthritis, various fevers and various pain symptoms.

Mammalian target of rapamycin (mTOR), as an important serine-threonine protein kinase downstream of PI3K/Akt, regulates the proliferation, survival, invasion and metastasis of tumor cells by activating ribosomal kinases. In recent years, the PI3K/AkUmTOR pathway has attracted much attention, and mTOR inhibitors have been proven to have considerable therapeutic prospects in a series of clinical studies targeting neuroendocrine tumors, kidney cancer, and breast cancer. and temsirolimus.

Contents of the invention

The present disclosure provides a pharmaceutical composition comprising a non-steroidal anti-inflammatory drug and an mTOR inhibitor. Also provided is the use of non-steroidal anti-inflammatory drugs and mTOR inhibitors in the preparation of medicines for treating or preventing viral hepatitis, especially for reducing the levels of HBV DNA, HBsAg and/or HBeAg.

The inventors unexpectedly found that the combination of non-steroidal anti-inflammatory drugs and mTOR inhibitors can produce synergistic effects, especially in reducing HBV DNA, HBsAg and/or HBeAg levels, which is stronger than any one of the two, and better than the two. or superimposed effect. The composition of the present disclosure is expected to achieve the effect of eliminating hepatitis B virus, and even achieve complete cure.

In one embodiment, the NSAIDs include aspirin, acetaminophen, indomethacin, naproxen, naproxen, diclofenac, ibuprofen, nimesulide, rofecoxib , at least one of celecoxib, the mTOR inhibitor includes at least one of rapamycin, everolimus and temsirolimus.

In one embodiment, the pharmaceutical composition is administered by a route selected from oral, rectal, nasal, pulmonary, topical, buccal and sublingual, vaginal, parenteral, subcutaneous, intramuscular, intravenous, dermal Intrathecal, intrathecal and epidural.

In one embodiment, the pharmaceutical composition is administered orally, preferably in the form of tablets or capsules.

The present disclosure also provides the use of non-steroidal anti-inflammatory drugs and mTOR inhibitors in the preparation of medicines for treating viral hepatitis.

According to some embodiments, the present disclosure provides the use of rapamycin or a pharmaceutically acceptable salt thereof and celecoxib or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating viral hepatitis.

The present disclosure further provides the use of non-steroidal anti-inflammatory drugs and mTOR inhibitors in the preparation of medicines for reducing HBV DNA, HBsAg and/or HBeAg levels.

According to some embodiments, the present disclosure also provides the use of celecoxib or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for reducing the level of HBV DNA, HBsAg and/or HBeAg.

The present disclosure provides a pharmaceutical composition comprising a non-steroidal anti-inflammatory drug and an mTOR inhibitor for use in the treatment of viral hepatitis. For example, the pharmaceutical composition includes rapamycin or a pharmaceutically acceptable salt thereof and celecoxib or a pharmaceutically acceptable salt thereof.

The present disclosure further provides a pharmaceutical composition comprising a non-steroidal anti-inflammatory drug and an mTOR inhibitor for reducing HBV DNA, HBsAg and/or HBeAg levels. For example, the pharmaceutical composition includes rapamycin or a pharmaceutically acceptable salt thereof and celecoxib or a pharmaceutically acceptable salt thereof.

In one embodiment, the mTOR inhibitor and the non-steroidal anti-inflammatory drug in the pharmaceutical composition or medicine of the present disclosure may exist independently in different dosage forms, or co-exist in one dosage form.

The present disclosure also provides a method of treating viral hepatitis using an mTOR inhibitor and a non-steroidal anti-inflammatory drug comprising administering a therapeutically effective amount of the mTOR inhibitor and the non-steroidal anti-inflammatory drug to an individual in need thereof. For example, the mTOR inhibitor is rapamycin or a pharmaceutically acceptable salt thereof, and the non-steroidal anti-inflammatory drug is celecoxib or a pharmaceutically acceptable salt thereof.

The present disclosure also provides a method of reducing HBV DNA, HBsAg and/or HBeAg levels using an mTOR inhibitor and a non-steroidal anti-inflammatory drug, comprising administering an mTOR inhibitor and a non-steroidal anti-inflammatory drug to a patient in need thereof individual. For example, the mTOR inhibitor is rapamycin or a pharmaceutically acceptable salt thereof, and the non-steroidal anti-inflammatory drug is celecoxib or a pharmaceutically acceptable salt thereof.

In one embodiment, the mTOR inhibitor and the non-steroidal anti-inflammatory drug may be administered separately, sequentially or simultaneously.

In one embodiment, the viral hepatitis is hepatitis B or hepatitis D, and the pharmaceutical composition is preferably used to reduce the levels of HBV DNA, HBsAg and/or HBeAg in patients with hepatitis B.

The technical solution of the present disclosure has the following beneficial effects:

1. The combination of non-steroidal anti-inflammatory drugs and mTOR inhibitors, especially celecoxib and rapamycin, is applied to the treatment or prevention of viral hepatitis, thereby providing a novel viral hepatitis treatment option.

2. The combination of non-steroidal anti-inflammatory drugs and mTOR inhibitors has a synergistic effect on effectively reducing the level of hepatitis B virus HBV DNA, HBsAg and/or HBeAg, and is expected to become a follow-up drug combination for the functional cure of hepatitis B .

3. As a common non-steroidal anti-inflammatory drug and mTOR inhibitor on the market, it has excellent clinical safety and pharmacokinetic properties, and has good druggability.

4. The combination of non-steroidal anti-inflammatory drugs and mTOR inhibitors can significantly improve the effect of removing hepatitis B virus, and has good synergistic effect.

Description of drawings

Figure 1: The inhibition results of HD042, HD082, and their combination on HBV DNA.

Figure 2: Inhibition results of HD042, HD082, and their combinations on HBeAg.

Figure 3: Results of HD042, HD082, and their combination for HBsAg inhibition.

Figure 4: Analysis of combined inhibitory effect of HD042 and HD082 on HBV DNA.

Figure 5: Analysis of combined inhibitory effects of HD042 and HD042 combination on HBeAg.

Figure 6: Analysis of combined inhibitory effect of HD042 and HD042 combination on HBsAg.

Figure 7: Detection results of the control compound RG7834.

Figure 8: ETV detection results of control compounds.

Detailed ways

The inventors of the present disclosure have screened out a composition with therapeutic effects on hepatitis B through multiple experiments, and further verified by biological experiments that the composition has the effect of potentially eliminating HBsAg and/or HBeAg, and is expected to functionally cure hepatitis B, eliminate Hepatitis B virus.

In one aspect, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of a non-steroidal anti-inflammatory drug and a therapeutically effective amount of an mTOR inhibitor, said composition being particularly useful in reducing HBV DNA, HBsAg and/or HBeAg levels.

In one embodiment, the NSAIDs include at least one of omeprazole, celecoxib, pantoprazole, rabeprazole, and esomeprazole, and the mTOR The inhibitor includes at least one of rapamycin, everolimus and temsirolimus.

In a preferred embodiment, the viral hepatitis is hepatitis B or hepatitis D.

In a preferred embodiment, the composition can reduce the level of hepatitis B virus HBV DNA, HBsAg and/or HBeAg.

In a preferred embodiment, the composition is administered by a route selected from the group consisting of: oral, rectal, nasal, pulmonary, topical, buccal and sublingual, vaginal, parenteral, subcutaneous, intramuscular, intravenous, dermal Intrathecal, intrathecal and epidural.

In a preferred embodiment, the composition is formulated for oral administration, preferably in the form of a tablet or capsule.

Definition of Terms

Rofecoxib and rapamycin herein include deuterated versions thereof.

viral hepatitis

The etiological classification of viral hepatitis has been recognized at present as five types of hepatitis viruses A, B, C, D, and E, respectively written as HAV, HBV, HCV, HDV, and HEV. Except that hepatitis B virus is a DNA virus, The rest are RNA viruses. Hepatitis caused by the above viruses are all viral hepatitis.

Hepatitis B is an infectious disease mainly caused by hepatitis B virus with liver lesions. Clinically, the main manifestations are loss of appetite, nausea, upper abdominal discomfort, pain in the liver area, and fatigue. Some patients may have jaundice, fever and hepatomegaly with liver function damage. Some patients can become chronic and even develop into liver cirrhosis, and a few can develop into liver cancer.

The pathogenic factor of hepatitis B virus is hepatitis B virus, abbreviated as HBV, and hepatitis B virus is a DNA virus. The genome is double-stranded, circular, partially closed DNA. The outermost layer of the virus is the outer membrane or coat of the virus, and the inner layer is the core part. The nucleoprotein is the core antigen (HBcAg), which cannot be detected in serum. In the serum of HBsAg-positive patients, three types of particles can be seen under the electron microscope, round and filamentous particles with a diameter of 22 nm, and less spherical particles with a diameter of 42 angstroms, also known as Dane’s particles, which are complete HBV particles .

The signs of hepatitis B are detected as follows: ① HBsAg and anti-HBs: HBsAg positive indicates that HBV is currently in the infection stage, and anti-HBs is positive for immune protective antibodies, indicating that immunity to HBV has been produced. Chronic HBsAg carriers are diagnosed on the basis of no clinical symptoms and signs, normal liver function, and HBsAg positive for more than 6 months. ②HBeAg and anti-HBe: HBeAg positive is an indicator of active HBV replication and strong infectivity. The change of tested serum from HBeAg positive to anti-HBe positive indicates that the disease has been relieved and the infectivity has weakened. ③HBcAg and anti-HBc: Positive HBcAg indicates the existence of complete HBV particle direct reaction, and the active replication of HBV is rarely used clinically due to the complicated detection method. Anti-HBc is a sign of HBV infection, and anti-HBc IgM positive indicates that it is in the early stage of infection and there is virus replication in the body. In chronic mild hepatitis B and HBsAg carriers, HBsAg, HBeAg and anti-HBc are all positive and highly contagious indicators are difficult to turn negative.

As used herein, "therapeutically effective amount" or "effective amount" refers to an amount effective at dosages and for periods of time required to achieve the desired therapeutic result. A therapeutically effective amount of a hepatitis B therapeutic will depend on the nature of the disorder or condition and on the particular agent, and can be determined by standard clinical techniques known to those skilled in the art.

The outcome of treatment can be, for example, reduction of symptoms, prolongation of survival, improvement of mobility, and the like. The result of treatment need not be a "cure." Treatment outcomes can also be preventative.

Route of administration

A medicament or pharmaceutical composition of the present disclosure is administered by any route appropriate for the condition to be treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) and the like. In certain embodiments, a medicament or pharmaceutical composition disclosed herein is administered by intravenous injection. It will be appreciated that the preferred route may vary depending, for example, on the condition of the recipient. One advantage of the disclosed medicaments or pharmaceutical compositions is that they are orally bioavailable and can be administered orally.

The pharmaceutical compositions of the present disclosure can be formulated with conventional carriers and excipients which will be selected according to ordinary practice. Tablets will contain excipients, glidants, fillers, binders, and the like. Aqueous formulations are prepared in sterile form and, when intended for delivery by parenteral administration, are generally isotonic. All formulations will optionally contain excipients such as those described in the "Handbook of Pharmaceutical Excipients" (1986).

Formulations include those suitable for the aforementioned routes of administration. Two or more drugs in the compositions of the present disclosure may be present in unit dosage form separately, or these drugs may be combined together to form a unit dosage form. Such formulations or dosage forms may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations are generally found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then shaping the product if necessary.

Formulations of the present disclosure suitable for oral administration may be presented as discrete units, such as capsules or tablets, each containing a predetermined amount of the active ingredient; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; or aqueous Oil-in-liquid emulsion or water-in-oil liquid emulsion. Compositions of the present disclosure may be in sustained or controlled release dosage forms. The composition may also be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.

Through the description of the following embodiments, the purpose, advantages and new features of the present disclosure will be more clear to those of ordinary skill in the art.

Example

Evaluation of synergistic effect of rapamycin and NSAIDs against HBV in vitro using HepG2-NTCP cells

1. Compound

Rapamycin (code HD082) and celecoxib (code HD042) were purchased from Shanghai Taosu Biotechnology Co., Ltd.

Compound preparation method is as follows:

Take the preparation of 20mM concentration as an example, the volume of solvent DMSO (μl) = sample mass (mg) × purity ÷ molecular weight ÷ 20 × 10 ⁶

The control compound was entecavir (ETV, batch number: P1214012; 99.0% purity), which was purchased from Shanghai Titan Technology Co., Ltd. The concentrations of the mother solutions of the above compounds were all 20 mM and stored at -20°C.

2. Cells and media

HepG2-NTCP cells: provided by Shanghai WuXi PharmaTech New Drug Development Co., Ltd.

Freezing PHH culture medium: mainly DMEM medium (Gibco product number 11960051) containing 10% fetal bovine serum (FBS, Hyclon product number SV3008703) and 1% penicillin-streptomycin, mainly used for cell culture.

Cryopreserved PHH plating medium: mainly containing 10% fetal bovine serum (FBS, Hyclon product number SV3008703) and 1% penicillin-streptomycin InvitroGRO CP Medium (BIOIVT product number S03316), mainly used for cell plating.

Virus infection medium: mainly containing 1% penicillin-streptomycin Williams' Medium E (SIGMA product number W1878), mainly used for HBV virus infection.

3. Main reagents

The main other reagents and cells used are listed in Table 1.

Table 1. Main Reagents and Cells

4. Experimental protocol

Plating Cells and Compound Treatment

On day 0, HepG2-NTCP cells were seeded into 48-well cell plates (7.5×10 ⁴ cells/well).

Infection virus and compound treatment

On the second day, the compound was added to pretreat the cells for 2 hours, and then type D HBV was added to infect the HepG2-NTCP cells (the compound was added at the same time as the infection). The test compound is set at 1-2 single-drug concentrations, and two-by-two combination medication is set, and the control compound is ETV and RG7834 with 3 concentrations each. Compound concentrations are shown in Table 2.

On the 3rd, 5th and 7th day, the medium containing the compound was replaced once. On day 9, cell supernatants were collected for detection of HBV DNA (qPCR), HBeAg and HBsAg (ELISA). After the cell supernatant was collected, CellTiter-Glo was added to detect cell viability, and the collected cells were cryopreserved (for later use).

Table 2. Test concentration of each compound

Sample testing

1) qPCR method to detect the content of HBV DNA in the cell culture supernatant

According to the instructions of QIAamp 96 DNA Blood Kit, extract the DNA in the cell culture supernatant. The content of HBV DNA was detected by qPCR method. PCR reaction: 95°C, 10min; 95°C, 15sec; 60°C, 1min, 40 cycles.

2) ELISA method to detect the content of HBeAg and HBsAg in the cell culture supernatant

The method refers to the kit instruction manual, and the method is briefly described as follows: take 50 μl of standard, sample and control substance and add them to the detection plate, then add 50 μl of enzyme conjugate to each well, incubate at 37°C for 60 minutes, wash the plate with washing solution and aspirate After drying, add 50 μl premixed luminescence substrate, incubate at room temperature in the dark for 10 minutes, and finally measure the luminescence value with a microplate reader.

CellTiter-Glo Cell Viability Assay

The cell viability was determined according to the instructions of the CellTiter-Glo kit. The method is briefly described as follows: After collecting the cell culture supernatant, add CellTiter-Glo (1:1 dilution in medium) to each well, incubate at room temperature for 10 minutes, and measure the luminescence value with a microplate reader.

data analysis

HBV DNA inhibition rate (%)=(1-the HBV copy number of compound group sample/the HBV copy number of DMSO control group)×100%

HBsAg inhibition rate (%)=(1-HBsAg value of sample/HBsAg value of DMSO control group)×100%

HBeAg inhibition rate (%)=(1-HBeAg value of sample/HBeAg value of DMSO control group)×100%

% cell viability=(signal value of sample-signal value of medium control)/(signal value of DMSO control-signal value of medium control)×100%

EC50 values were calculated using GraphPad Prism software (four parameter logistic equations).

The Combination Index (CI) software CompuSyn software V1.0. software was used to analyze the synergistic effect of rapamycin and celecoxib with the Non-Constant Combo method.

Experiment Accepted Metrics

If the control compound test in the experiment yields the expected results, the other data in the experiment are considered to be valid and available. And the data will be analyzed and explained in the report.

Result analysis

See Table 3-6 and Figure 1-6 for the test results. When rapamycin and celecoxib are used in combination, they have an obvious synergistic effect on inhibiting HBV DNA, HBsAg and HBeAg.

In order to analyze the synergistic effect more clearly, a series of data were analyzed using Combination Index (CI) software CompuSyn software V1.0. The results are shown in Table 4-6 and Figure 4-6.

The results of the reference compound ETV are shown in Table 5.

Table 3 The inhibitory effects of HD042 and HD082 on HBV respectively

Table 4 Analysis of combined effect of HD042+HD082 on HBV DNA inhibition rate

Table 5 Analysis of combined effect of HD042+HD082 on HBeAg inhibition rate

Table 6 Analysis of combined effect of HD042+HD082 on HBsAg inhibition rate

The above test results show that when HD042 and HD082 are used in combination, they can effectively reduce HBV DNA, HBsAg and/or HBeAg, and have obvious synergistic effects.

According to the above results, the combination of rapamycin and celecoxib can produce a synergistic effect and is expected to become a new combination of hepatitis B drugs.

Although the present disclosure has been described with reference to specific embodiments, those skilled in the art will recognize that changes or improvements may be made to the described embodiments without departing from the spirit and scope of the present disclosure, all of which are described in this application. within the scope of protection.

Claims

A pharmaceutical composition comprising a non-steroidal anti-inflammatory drug and an mTOR inhibitor.
The pharmaceutical composition according to claim 1, wherein the non-steroidal anti-inflammatory drug is selected from aspirin, acetaminophen, indomethacin, naproxen, naproxen, diclofenac, ibuprofen, nimesulide , at least one of rofecoxib and celecoxib; the mTOR inhibitor is selected from at least one of rapamycin or a pharmaceutically acceptable salt thereof, everolimus and temsirolimus.
The pharmaceutical composition according to claim 1 or 2, wherein the non-steroidal anti-inflammatory drug is celecoxib or a pharmaceutically acceptable salt thereof, and the mTOR inhibitor is rapamycin or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition of any one of claims 1-3, wherein the pharmaceutical composition is a pharmaceutical composition selected from the group consisting of oral, rectal, nasal, pulmonary, topical, buccal and sublingual, Vaginal, parenteral, subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural.
The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is an oral dosage form.
The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is a tablet or a capsule.
Use of non-steroidal anti-inflammatory drugs and mTOR inhibitors in the preparation of medicines for treating viral hepatitis.
The use according to claim 7, wherein the viral hepatitis is hepatitis B.
The use according to claim 7 or 8, wherein the non-steroidal anti-inflammatory drug is celecoxib or a pharmaceutically acceptable salt thereof, and the mTOR inhibitor is rapamycin or a pharmaceutically acceptable salt thereof.
Use of non-steroidal anti-inflammatory drugs and mTOR inhibitors in the preparation of medicines for reducing HBV DNA, HBsAg and/or HBeAg levels.
The use according to claim 10, wherein the non-steroidal anti-inflammatory drug is celecoxib or a pharmaceutically acceptable salt thereof, and the mTOR inhibitor is rapamycin or a pharmaceutically acceptable salt thereof.
The use according to claim 10 or 11, wherein the use is to reduce the level of HBV DNA, HBsAg and/or HBeAg in patients with hepatitis B.