CN114209844B - A pharmaceutical composition for treating viral hepatitis - Google Patents

A pharmaceutical composition for treating viral hepatitis Download PDF

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CN114209844B
CN114209844B CN202210101654.8A CN202210101654A CN114209844B CN 114209844 B CN114209844 B CN 114209844B CN 202210101654 A CN202210101654 A CN 202210101654A CN 114209844 B CN114209844 B CN 114209844B
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CN114209844A (en
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李瑛颖
陈明键
仇思念
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China Israel Hyde Artificial Intelligence Drug Research And Development Co ltd
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses

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Abstract

The application provides a pharmaceutical composition for treating viral hepatitis, which comprises a proton pump inhibitor and an mTOR inhibitor, and application thereof in treating or preventing viral hepatitis, and application thereof in preparing medicaments for treating or preventing viral hepatitis, particularly hepatitis B.

Description

A pharmaceutical composition for treating viral hepatitis
Technical Field
The application relates to the technical field of antiviral drugs, in particular to a compound for treating or preventing viral hepatitis, a pharmaceutical composition and application thereof.
Background
Human Hepatitis B Virus (HBV) infection is a major public health problem worldwide. After acute hepatitis B virus infection, about 8% of hepatitis B virus still develops into chronic hepatitis B infection, and persistent HBV infection can cause cirrhosis and even liver cancer. The hepatitis B transmission pathway is mainly through vertical transmission and horizontal transmission. Vertical transmission refers to mother-to-baby transmission; horizontal transmission is primarily through the blood.
The treatment of hepatitis b is also a long-term process, and the treatment aims to inhibit or eliminate HBV to the maximum extent, relieve inflammation and necrosis of liver cells and liver fibrosis, delay and stop the progress of diseases, and reduce and prevent the occurrence of liver decompensation, liver cirrhosis, HCC and complications thereof, thereby improving the quality of life and prolonging the survival time.
There are many hepatitis b therapeutic agents currently on the market, mainly by antiviral treatment using interferon or nucleoside analogs. In the case of interferon, recombinant DNA leukocyte interferon (IFN-. Alpha.) inhibits the replication of HBV. However, when the interferon is used for treating hepatitis B, strong adverse reactions are often accompanied, including bone marrow suppression, thyroid function influence, depression and the like.
Nucleoside analogues inhibit HBV production primarily by inhibiting reverse transcriptase activity during HBV replication, and clinically useful drugs include the following classes: lamivudine and famciclovir, such as acyclovir, adefovir, entecavir, tenofovir, foscarnet and the like, and the medicaments have certain HBV inhibiting effect.
Although these reverse transcriptase inhibitors can effectively reduce HBV DNA level and make patients control HBV level, they have no direct effect on the clearance of HBeAg and HBsAg because their target of action is the process of reverse transcription of RNA into DNA. Therefore, the seroconversion probability of HBeAg and HBsAg in the single-drug treatment of the nucleoside analogue is extremely low, hepatitis B cannot be really cured, and patients need to take the drugs for a long time or even for life.
Although the reverse transcriptase inhibitor can control the level of hepatitis B virus, the problems of drug resistance, huge medical cost, serious side effects of the drug and the like are not small. The key point is that no medicine can completely eliminate viruses to functionally cure hepatitis B at present. Therefore, there is an urgent need in the art to provide a new drug for treating hepatitis b that achieves a functional cure without the need for long-term or even lifelong administration.
Lansoprazole (Lansoprazole) is a novel drug for inhibiting gastric acid secretion, has obvious inhibition effect on basic gastric acid secretion and gastric acid secretion caused by all stimulators (such as histamine, carbamoylcholine and the like), has the acid inhibition effect obviously superior to that of an H2 receptor blocker, and has obvious dependence on the inhibition degree and the concentration. Lansoprazole can be rapidly transformed into sulfenic acid and sulfenic derivatives through parietal cell membranes under an acidic condition to exert drug effect, has an inhibition effect on helicobacter pylori (Hp), and is 4 times of that of omeprazole. The traditional Chinese medicine composition is clinically used for treating duodenal ulcer, gastric ulcer, anastomotic oral ulcer, reflux esophagitis and Zollinger-Ellison syndrome, and has a remarkable curative effect.
Mammalian target of rapamycin (mTOR), an important serine-threonine protein kinase downstream of PI3K/Akt, regulates tumor cell proliferation, survival and invasive metastasis by activating ribosomal kinases. In recent years, the PI3K/AkUmTOR pathway is of great interest, and mTOR inhibitors have proved to have considerable treatment prospects in a series of clinical researches on neuroendocrine tumors, kidney cancers, breast cancers and the like, and represent drugs of rapamycin, everolimus and temsirolimus.
Disclosure of Invention
The present application provides a pharmaceutical composition comprising a proton pump inhibitor, and an mTOR inhibitor, and its use for the preparation of a medicament for the treatment or prevention of viral hepatitis, in particular for reducing HBsAg and/or HBeAg levels.
The inventors have surprisingly found that the combination of a proton pump inhibitor and an mTOR inhibitor produces a synergistic effect, particularly in reducing HBsAg and/or HBeAg levels, which is stronger than either and better than the additive effect. The composition of the application hopefully achieves the effect of eliminating hepatitis B virus and even achieves complete cure.
In one embodiment, the proton pump inhibitor comprises at least one of omeprazole, lansoprazole, pantoprazole, rabeprazole, esomeprazole, and the mTOR inhibitor comprises at least one of rapamycin, everolimus, and temsirolimus.
In one embodiment, the pharmaceutical 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, intradermal, intrathecal and epidural.
In one embodiment, the pharmaceutical composition is administered orally, preferably in the form of a tablet or capsule.
The application also provides application of the pharmaceutical composition in preparing a medicament for treating viral hepatitis.
The application also provides application of the lansoprazole or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof in preparing a medicament for reducing the level of HBV DNA, HBsAg and/or HBeAg.
In one embodiment, the viral hepatitis is hepatitis b or hepatitis d, and the use is preferably the use of reducing the level of HBsAg and/or HBeAg in a hepatitis b patient.
In one embodiment, the present application provides the use of lansoprazole, or a pharmaceutically acceptable salt thereof, and rapamycin, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating hepatitis b.
The technical scheme of the application has the following beneficial effects:
1. the use of a combination of a proton pump inhibitor and an mTOR inhibitor, particularly lansoprazole and rapamycin, for the treatment or prevention of viral hepatitis provides a novel viral hepatitis treatment option.
2. The composition of the proton pump inhibitor and the rapamycin has synergistic effect on effectively reducing the level of HBsAg and/or HBeAg of hepatitis B virus, and is expected to become a subsequent medicine composition for functional cure of hepatitis B.
3. As a common proton pump inhibitor and an mTOR inhibitor in the market, the compound has excellent clinical safety and pharmacokinetic property and better druggability.
4. The composition of the proton pump inhibitor and the mTOR inhibitor can obviously improve the effect of eliminating hepatitis B virus and has better synergy.
Description of the drawings:
FIG. 1: inhibition results of HD017, HD082, and combinations thereof on HBeAg.
FIG. 2 is a schematic diagram: HD017, HD082, and combinations thereof on HBsAg inhibition results.
FIG. 3: the combination of HD017 and HD082 was analyzed for the combined inhibitory effect of HBeAg.
FIG. 4 is a schematic view of: the combination of HD017 and HD082 was analyzed for combined inhibition of HBsAg.
FIG. 5: the result of the test was the control compound RG 7834.
FIG. 6: ETV test results for control compound.
Detailed Description
The inventor screens out the composition with the hepatitis B treatment effect through multiple experiments, and further verifies through biological experiments that the composition has the effect of potentially removing HBsAg and/or HBeAg, is expected to functionally cure hepatitis B and remove hepatitis B virus.
In one aspect, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a proton pump inhibitor, and a therapeutically effective amount of an mTOR inhibitor, and its use in medicaments for the treatment or prevention of viral hepatitis, in particular for reducing HBsAg and/or HBeAg levels.
In one embodiment, the proton pump inhibitor comprises at least one of omeprazole, lansoprazole, pantoprazole, rabeprazole, esomeprazole, and the mTOR inhibitor comprises 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 is capable of reducing the level of hepatitis b virus HBsAg and/or HBeAg.
In a preferred embodiment, the composition is formulated for administration by a route selected from the group consisting of: oral, rectal, nasal, pulmonary, topical, buccal and sublingual, vaginal, parenteral, subcutaneous, intramuscular, intravenous, intradermal, 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
Lansoprazole is a novel drug for inhibiting gastric acid secretion, has obvious inhibition effect on basic gastric acid secretion and gastric acid secretion caused by all stimulators (such as histamine, carbamoylcholine and the like), has the inhibition effect obviously superior to that of an H2 receptor blocker, and has obvious dependence on the inhibition degree and concentration. Lansoprazole can rapidly pass through parietal cell membrane under acidic condition and is converted into sulfenic acid and sulfenic derivatives to exert drug effect, and has inhibitory effect on helicobacter pylori (Hp) 4 times that of omeprazole. The traditional Chinese medicine composition is clinically used for treating duodenal ulcer, gastric ulcer, anastomotic oral ulcer, reflux esophagitis and Zollinger-Ellison syndrome, and has obvious curative effect.
Mammalian target of rapamycin (mTOR), an important serine-threonine protein kinase downstream of PI3K/Akt, regulates the proliferation, survival and invasive metastasis of tumor cells by activating ribosomal kinases. In recent years, the PI3K/AkUmTOR pathway is concerned, and mTOR inhibitors are proved to have considerable treatment prospects in a series of clinical researches on neuroendocrine tumors, kidney cancers, breast cancers and the like, and represent the drugs of rapamycin, everolimus and temsirolimus.
Lansoprazole and rofecoxib herein include deuterated versions thereof.
Viral hepatitis
The etiological typing of viral hepatitis is currently recognized by five hepatitis viruses, namely hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus and hepatitis E virus, which are respectively written as HAV, HBV, HCV, HDV and HEV, and the rest are RNA viruses except the hepatitis B virus which is a DNA virus.
Hepatitis b is an infectious disease mainly caused by hepatitis b virus, and is a liver disease. Clinically, anorexia, nausea, epigastric discomfort, liver pain, and asthenia are the main manifestations. Some patients may have jaundice fever and hepatomegaly with impaired liver function. Some patients can become chronic, even develop cirrhosis of the liver, and a few can develop liver cancer.
The etiological agent of hepatitis b is hepatitis b virus, abbreviated as HBV, and hepatitis b virus is DNA virus. The genome is double-stranded, circular, incompletely closed DNA. The outermost layer of the virus is the outer membrane or coat membrane of the virus, the inner layer is the core part, and the nucleoprotein is the core antigen (HBcAg) and cannot be detected in the serum. Serum from HBsAg positive patients was observed under electron microscope to show 3 kinds of particles, circular and filamentous particles with a diameter of 22nm, and less spherical particles with a diameter of 42 angstroms, also called Dane's particles, as complete HBV particles.
The markers for hepatitis b were detected as follows: (1) HBsAg and anti-HBs: HBsAg positive indicates that HBV is currently in the stage of infection, and anti-HBs positive for immunoprotective antibodies indicates that immunity to HBV has developed. The diagnosis of chronic HBsAg carrier is based on no clinical symptoms and signs, normal liver function, and HBsAg positive lasting more than 6 months. (2) HBeAg and anti-HBe: HBeAg positive is an index of HBV active replication and strong infectivity, and the change of the detected serum from HBeAg positive to anti-HBe positive indicates that the disease has remission and weakened infectivity. (3) HBcAg and anti-HBc: HBcAg positive suggests that there is a direct reaction of complete HBV particles, and active replication of HBV is less clinically useful due to the complex detection method. anti-HBc is a marker of HBV infection, and anti-HBc IgM positive indicates that the virus is replicated in vivo at an early stage of infection. HBsAg, HBeAg and anti-HBc are all positive in chronic mild hepatitis B and HBsAg carriers, and have high infectivity index and are difficult to convert from negative to positive.
As used herein, "therapeutically effective amount" or "effective amount" refers to an amount that is effective at a dose and for a period of time required to achieve a desired therapeutic result. A therapeutically effective amount of a therapeutic agent for hepatitis b 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 therapeutic result may be, for example, alleviation of symptoms, prolongation of survival, increased mobility, and the like. The therapeutic result need not be a "cure". The therapeutic outcome may also be prophylactic.
Route of administration
The medicaments or pharmaceutical compositions of the present disclosure are administered by any route suitable 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, the medicament or pharmaceutical composition disclosed herein is administered by intravenous injection. It will be appreciated that the preferred route may vary depending on, for example, 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.
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, intradermal, intrathecal and epidural.
In a preferred embodiment, the composition is administered orally, preferably in the form of a tablet or capsule.
The pharmaceutical compositions of the present disclosure may be formulated with conventional carriers and excipients, which will be selected in accordance with common practice. Tablets will contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in sterile form and, when used for delivery by non-oral administration, are generally isotonic. All formulations will optionally contain Excipients such as those described in the Handbook of Pharmaceutical Excipients (1986).
The formulations include those suitable for the aforementioned routes of administration. The two or more agents of the compositions of the present application may be present separately in unit dosage form or the agents may be combined together to form a unit dosage form. The formulations or dosage forms may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations are commonly 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 is composed of 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 as necessary.
Formulations of the present application suitable for oral administration may exist as follows: discrete units, such as capsules or tablets, each containing a predetermined amount of active ingredient; a powder or granules; solutions or suspensions in aqueous or non-aqueous liquids; or an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The pharmaceutical compositions of the present disclosure may be sustained or controlled release formulations. The pharmaceutical compositions of the present disclosure may also be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.
Objects, advantages and novel features of the present application will become apparent to one of ordinary skill in the art from the following description of the embodiments.
Examples
HepG2-NTCP cells were used to evaluate the in vitro anti-HBV synergy of rapamycin and proton pump inhibitors.
1. Compound (I)
Rapamycin (No. HD 082) and lansoprazole (No. HD 017) were purchased from shanghai ceramic biotechnology limited.
The compound preparation method comprises the following steps:
in the case of preparation of 20mM concentration, volume (. Mu.l) = sample mass (. Mu.l). Times.purity ÷ molecular weight ÷ 20X 10 of DMSO as solvent 6
The control compound was entecavir (ETV, lot: P1214012;99.0% purity) purchased from Shanghai Tantake Technology, inc. The mother liquors of the above compounds were all at 20mM concentration and stored at-20 ℃.
2. Cells and culture media
HepG2-NTCP cells: provided by Shanghai Mingkude New drug development Co., ltd
Freezing and storing a PHH culture medium: mainly a DMEM medium (Gibco cat No. 11960051) containing 10% fetal bovine serum (FBS, hyclone cat No. SV 3008703) and 1% penicillin-streptomycin, mainly used for the culture of cells.
Freezing and storing PHH plating culture medium: mainly contains 10% of fetal bovine serum (FBS, hyclon cat # SV 3008703) and 1% of penicillin-streptomycin InvitroGRO CP Medium (BIOIVT cat # S03316), and is mainly used for cell plating.
Virus infection medium: mainly contains 1% penicillin-streptomycin Williams' Medium E (SIGMA goods number W1878), and is mainly used for HBV virus infection.
3. Primary reagent
The main other reagents and cells used are shown in table 1.
TABLE 1 Primary reagents and cells
Figure BDA0003492657550000081
Figure BDA0003492657550000091
4. Experimental protocol
Plating cells and compound treatment
Day 0HepG2-NTCP cells were seeded in 48-well plates (7.5X 10) 4 Cells/well).
Infectious virus and compound treatment
On day 2, cells were pretreated with the compound for 2 hours before the addition of D-type HBV to infect HepG2-NTCP cells (compound was added at the same time as infection). Test compounds were set at 3 single drug concentrations and combinations were set two by two, control compounds were ETV and RG7834 at 3 concentrations each. The compound concentrations are shown in Table 2.
The culture medium containing the compound was changed once on day 3, day 5 and day 7. On day 9, cell supernatants were collected for detection of HBV DNA (qPCR), HBeAg and HBsAg (ELISA). After collecting the cell supernatant, cellTiter-Glo was added to test cell viability, and the collected cells were stored frozen (for future use).
TABLE 2 test concentrations of the respective Compounds
Figure BDA0003492657550000092
Sample detection
1) qPCR method for detecting HBV DNA content in cell culture supernatant
DNA was extracted from the cell culture supernatant according to the QIAamp 96DNA Blood Kit instructions. The content of HBV DNA was detected by qPCR method. And (3) PCR reaction: at 95 ℃ for 10min;95 ℃ for 15sec; at 60 ℃ for 1min,40 cycles.
2) ELISA method for detecting content of HBeAg and HBsAg in cell culture supernatant
The method refers to the kit specification, and the method is briefly described as follows: respectively adding 50 mu l of standard substance, sample and reference substance into a detection plate, then adding 50 mu l of enzyme conjugate into each hole, incubating for 60 minutes at 37 ℃, washing the plate with washing liquor, then sucking dry, then adding 50 mu l of premixed luminescent substrate, incubating for 10 minutes at room temperature in a dark place, and finally measuring the luminescent value by an enzyme-linked immunosorbent assay.
CellTiter-Glo cell viability assay
Cell viability was determined with reference to CellTiter-Glo kit instructions, the method is briefly as follows: after collecting the cell culture supernatant, cellTiter-Glo (medium 1.
Data analysis
HBV DNA inhibition (%) = (1-HBV copy number of compound group sample/HBV copy number of DMSO control group) × 100%
HBsAg inhibition (%) = (1-HBsAg value of sample/DMSO control HBsAg value) × 100%
HBeAg inhibition (%) = (1-HBeAg value of sample/DMSO control HBeAg value) × 100%
% cell viability = (signal value of sample-signal value of media control)/(signal value of DMSO control-signal value of media control) × 100%
EC50 values were calculated using GraphPad Prism software (four parameter logistic equations).
Combination Index (CI) software CompuSyn software V1.0 software was used to analyze the synergy of rapamycin and lansoprazole using the Non-Constant Combo method.
Index of experiment acceptance
Other data in an experiment are considered valid if the control compound tested in the experiment gave the expected result. And the data will be analyzed and accounted for in the report.
Analysis of results
The detection results are shown in tables 3-5 and fig. 1-4, and when the rapamycin and the lansoprazole are combined, the rapamycin and the lansoprazole have obvious synergistic effect on the aspect of inhibiting the HBsAg and the HBeAg.
To analyze their synergy more clearly, a series of data were analyzed using the Combination Index (CI) software CompuSyn software V1.0. Software, see tables 4 and 5, FIGS. 3 and 4.
TABLE 3 inhibitory Effect of HD017 and HD082 on HBV, respectively
Figure BDA0003492657550000111
TABLE 4 analysis of combined effect of HD017+HD082 on HBeAg inhibition rate
Figure BDA0003492657550000112
Figure BDA0003492657550000121
TABLE 5 analysis of combined effect of HD017 Bu HD082 on HBsAg inhibition rate
Figure BDA0003492657550000122
The test results show that when HD017 and HD082 are used together, HBsAg and/or HBeAg can be effectively reduced, and obvious synergistic effect is achieved.
According to the results, the combination of the rapamycin and the lansoprazole can generate a synergistic effect, and is expected to become a novel drug combination for treating hepatitis B.
While the present application has been described with reference to particular embodiments, those skilled in the art will recognize that changes or modifications can be made to the described embodiments without departing from the spirit and scope of the present application, which is defined by the appended claims.

Claims (6)

1. Use of a pharmaceutical composition in the manufacture of a medicament for the treatment of hepatitis b, wherein the pharmaceutical composition comprises lansoprazole, or a pharmaceutically acceptable salt thereof, and rapamycin, or a pharmaceutically acceptable salt thereof.
2. Use of lansoprazole, or a pharmaceutically acceptable salt thereof, in combination with rapamycin, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for reducing the level of HBV DNA, HBsAg and/or HBeAg in a hepatitis B patient.
3. The use according to claim 1, wherein the use is for reducing the level of HBsAg and/or HBeAg in a hepatitis B patient.
4. The use of any one of claims 1-3, wherein the medicament is a medicament administered by a route selected from the group consisting of: oral and parenteral.
5. The use of claim 4, wherein the medicament is an oral dosage form.
6. The use of claim 5, wherein the medicament is a tablet or capsule.
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