CN114224888B - Application of lansoprazole in preparing medicine for treating or preventing viral hepatitis - Google Patents

Abstract

The invention provides application of a compound 1 in preparing a medicament for treating or preventing viral hepatitis. Specifically, the invention provides the application of the compound 1, the derivative thereof or the pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing viral hepatitis, preferably the viral hepatitis is hepatitis B. The present invention also provides a pharmaceutical composition for treating or preventing viral hepatitis, comprising compound 1 or a pharmaceutically acceptable salt thereof and optionally one or more additional therapeutic or prophylactic agents, and a pharmaceutically acceptable carrier.

Description

Application of lansoprazole in preparing medicine for treating or preventing viral hepatitis

Technical Field

The invention relates to the technical field of antiviral drugs, and in particular relates to a pharmaceutical composition for treating or preventing viral hepatitis 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 therapeutic goals are to suppress or eliminate HBV to the maximum extent, to alleviate hepatocyte inflammatory necrosis and liver fibrosis, to delay and arrest disease progression, and to reduce and prevent liver decompensation, cirrhosis, HCC and its complications, thereby improving quality of life and prolonging survival time.

There are many hepatitis b therapeutic drugs on the market today, mainly by antiviral treatment with interferon or nucleoside analogues. 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 the level of HBVDNA and control the level of hepatitis B virus in patients, they have no direct effect on the clearance of HBeAg and HBsAg because the 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 at present, no medicine can completely eliminate viruses to achieve the functional cure of hepatitis B. Therefore, the urgent need in the art is to provide a new drug for treating hepatitis b, which can eliminate HBsAg and achieve a functional cure.

Lansoprazole can be used to treat gastric ulcer or reflux esophagitis and other conditions caused by gastric hyperacidity. It can inhibit the action of a protein called ATPase (ATPase) with catalytic action in gastric parietal cells, so that the last step of gastric acid production is hindered, and finally, the production of gastric acid is directly reduced and the stimulation of the stomach or esophagus is reduced, so that the ulcer part is gradually rested and recovered. The composition can also be administered with other antibiotics to treat gastric ulcers caused by H.pyrori bacterial infection.

At present, the research aiming at the lansoprazole indication mainly aims at the application of the lansoprazole indication in treating or preventing diseases such as gastric ulcer and the like, and no research report is available for treating or preventing viral hepatitis.

Disclosure of Invention

According to the invention, a series of compounds are screened out by an artificial intelligence system based on a plurality of target spots and big data analysis, wherein the compounds comprise a compound 1 with hepatitis B treatment effect, and further biological experiment verification proves that the compound 1-lansoprazole has the effect of removing HBsAg and HBeAg, and is expected to functionally cure hepatitis B and remove hepatitis B virus.

The present invention provides a novel viral hepatitis treatment option by applying compound 1, i.e., lansoprazole, a derivative thereof, or a pharmaceutically acceptable salt thereof, to the treatment or prevention of viral hepatitis.

In one aspect, the invention provides the use of lansoprazole, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prevention of viral hepatitis.

In a preferred embodiment, the pharmaceutically acceptable salt is selected from the group consisting of acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, malate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, thiocyanate, tosylate, undecanoate, sodium, calcium, potassium, ammonium, tetraethylammonium, methylammonium, dimethylammonium, and ethanolamine salts, preferably lansoprazole glucuronide monosodium salt.

In a preferred embodiment, the viral hepatitis is hepatitis b or hepatitis d.

In a preferred embodiment, the medicament is capable of reducing Hepatitis B Virus (HBV) load, HBsAg and/or HBeAg levels.

In a preferred embodiment, the medicament further comprises one or more additional therapeutic or prophylactic agents.

In a preferred embodiment, the additional therapeutic or prophylactic agent is selected from at least one of an interferon, a pegylated interferon, or a nucleoside analogue, preferably the nucleoside analogue is selected from entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide.

In a preferred embodiment, the nucleoside analogue is selected from entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide.

In a preferred embodiment, the medicament 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 medicament is formulated for oral administration, preferably in the form of a tablet or capsule, more preferably an enteric coated tablet.

In another aspect, the invention provides a pharmaceutical composition for treating or preventing viral hepatitis comprising a therapeutically effective amount of lansoprazole, or a pharmaceutically acceptable salt thereof, and optionally one or more additional therapeutic or prophylactic agents, and a pharmaceutically acceptable carrier.

The technical scheme of the invention has the following beneficial effects:

1. lansoprazole, or a pharmaceutically acceptable salt thereof, is used for treating or preventing viral hepatitis, thereby providing a novel viral hepatitis treatment option.

2. Lansoprazole or pharmaceutically acceptable salt thereof can effectively reduce the load of Hepatitis B Virus (HBV), the HBsAg level and/or the HBeAg level, has a wide application prospect, and particularly can reduce the technical effect of the HBsAg level and/or the HBeAg level, so that the functional cure of hepatitis B becomes possible.

3. The lansoprazole or pharmaceutically acceptable salt thereof has excellent clinical safety and pharmacokinetic property, and has better drug forming property.

4. Lansoprazole, or a pharmaceutically acceptable salt thereof, can be optionally combined with one or more additional therapeutic or prophylactic agents, particularly drugs that lower viral titers but do not completely eliminate the virus and do not reduce HBsAg and/or HBeAg levels, eliminates hepatitis b virus from different aspects, with the potential for synergy.

Description of the drawings:

FIG. 1: the combination of lansoprazole with different concentrations, 0.1nM entecavir, 20 μ M lansoprazole and 0.1nM entecavir has the effect of inhibiting HBVDNA, HBsAg and HBeAg.

Detailed Description

According to the invention, through an artificial intelligence system, based on a plurality of hepatitis B treatment targets and big data analysis, the compound 1 (i.e. lansoprazole) with the hepatitis B treatment effect is screened out, and further through verification of a biological experiment, the compound 1 with the effect of removing HBsAg and HBeAg is obtained, so that the hepatitis B virus can be cured functionally and removed.

In one aspect, the present invention provides the use of lansoprazole, a derivative thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment or prevention of viral hepatitis.

In a preferred embodiment, the derivatives of lansoprazole include deuterated lansoprazole, e.g., compounds 1-2, compounds 1-3, or lansoprazole deuterated at other sites, e.g., methyl, phenyl ring, etc. To those skilled in the art, deuterated compounds do not change the original properties of the compound, but can slow metabolic processes, thereby extending half-life.

Can more effectively play the role of the medicine.

In a preferred embodiment, the pharmaceutically acceptable salts are lansoprazole sulfonate and lansoprazole sodium salt. In a preferred embodiment, the viral hepatitis is hepatitis b. In a preferred embodiment, the medicament is capable of reducing Hepatitis B Virus (HBV) load, HBsAg and/or HBeAg levels.

Lansoprazole can be used to treat gastric ulcer or reflux esophagitis and other conditions caused by gastric hyperacidity. It can inhibit the action of ATP enzyme (ATPase) which is a protein with catalytic action in the cell of gastric wall, so that the last step of gastric acid production is hindered, and the generation of gastric acid is directly reduced, and the stimulation of stomach or esophagus is reduced, so that the ulcer part can be gradually rested and recovered. The composition can also be administered with other antibiotics to treat gastric ulcers caused by H. However, there have been no reports of its use in the treatment of hepatitis B, let alone its ability to reduce HBsAg and/or HBeAg levels.

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, the symptoms of anorexia, nausea, epigastric discomfort, liver pain and hypodynamia are mainly manifested. 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, which is DNA virus. The genome is a 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 have 3 kinds of particles, circular and filamentous particles with a diameter of 22nm, and a smaller number of particles with a diameter of 42 nm. The spherical particles of (a), also known as Dane's particles, are intact 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 basis of the chronic HBsAg carrier is that the chronic HBsAg carrier has no clinical symptoms and physical signs, the liver function is normal, and the HBsAg is continuously positive for 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 indicates that complete HBV particle direct reaction exists, and HBV active replication is less clinically used due to the complex detection method. anti-HBc is a marker of HBV infection, and anti-HBcIgM positive indicates that in early infection, there is virus replication in vivo. 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.

In a preferred embodiment, the medicament further comprises one or more additional therapeutic or prophylactic agents. In a preferred embodiment, the additional therapeutic or prophylactic agent is selected from an interferon or a nucleoside analogue. In a preferred embodiment, the nucleoside analogue is selected from entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide.

Additional therapeutic or prophylactic agents

In some embodiments, the additional therapeutic or prophylactic agent is selected from one or more of entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide, for example, one selected from entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide or at least two selected from entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide.

Entecavir (Entecavir) is chemically known as 2-amino-1, 9-dihydro-9- [ (1s, 3r, 4s) -4-hydroxy-3- (hydroxymethyl) -2-methylenecyclopentane ] -6H-purin-6-one and has the following structural formula:

US patent US5206244 discloses entecavir and its use for the treatment of hepatitis b virus; a novel method for synthesizing entecavir is disclosed in WO 9809964; WO0164421 discloses low dose entecavir solid formulations.

Entecavir is a highly potent antiviral agent developed in the 90's 20 th century by schrobo, usa and has a strong anti-HBV effect. It can be phosphorylated to active triphosphate, which has a half-life in cells of 15h. Entecavir triphosphate inhibits all three activities of the viral polymerase (reverse transcriptase) by competing with deoxyguanosine triphosphate, the natural substrate of HBV polymerase: (1) the initiation of HBV polymerase; (2) formation of a reverse transcribed minus strand of the pregenomic mRNA; and (3) synthesis of HBVDNA positive strand.

Tenofovir disoproxil fumarate (the name of England: tenofovirdosisoproximate, TDF; the chemical name is (R) - [ [2- (6-amino-9H-purin-9-yl) -1-methylethoxy ] methyl ] phosphonic acid diisopropoxycarbonyl methyl ester fumarate) is an ester precursor of tenofovir, belongs to a novel nucleotide reverse transcriptase inhibitor, and has the activity of inhibiting HBV viruses.

TDF is another novel open-ring nucleoside phosphonate successfully developed by Gilidder company in the United states after Adefovir dipivoxil, is first marketed in the United states in 10 months in 2001, and is now marketed in Europe, australia, canada, and the like.

TDF inhibits viral polymerase in vivo by competitively binding to the natural deoxyribose substrate and terminates DNA strand synthesis by insertion into DNA. The main action mechanism is that the tenofovir is hydrolyzed into tenofovir after being taken orally, the tenofovir is phosphorylated by cell kinase to generate a metabolite tenofovir diphosphate with pharmacological activity, the tenofovir diphosphate competes with 5 '-deoxyadenosine triphosphate to participate in the synthesis of virus DNA, and after entering the virus DNA, the DNA is prevented from being prolonged due to the lack of 3' -OH groups, so that the replication of the virus is blocked. Clinical application shows that TDF has obvious curative effect on HBV virus and less toxic side effect, so that TDF has wide clinical application foreground.

Tenofovir alafenamide (Tenofovir arlafenamide), a prodrug of the new Nucleoside Reverse Transcriptase Inhibitor (NRTI) Tenofovir (Tenofovir), developed by Gilidard scientific, USA. Compared with the prior generation of similar anti-hepatitis B medicine tenofovir disoproxil TDF, the antiviral activity of tenofovir alafenamide is 10 times, the stability in blood plasma is 200 times, and the half-life period is improved by 225 times. Compared with TDF, tenofovir alafenamide requires only one-tenth of the dose of TDF administered to achieve the same antiviral efficacy as TDF. Therefore, the tenofovir alafenamide is used for preventing or/and treating Hepatitis B Virus (HBV) infection and has better curative effect, higher safety and lower drug resistance.

In addition to the above active agents, the medicaments or pharmaceutical compositions described herein may optionally further comprise one or more additional other agents useful in the treatment of HBV, such as, but not limited to, 3-dioxygenase (IDO) inhibitors, antisense oligonucleotides targeted to viral mRNA, apolipoprotein A1 modulators, arginase inhibitors, B-and T-lymphocyte attenuating agent inhibitors, bruton's Tyrosine Kinase (BTK) inhibitors, CCR2 chemokine antagonists, CD137 inhibitors, CD160 inhibitors, CD305 inhibitors, CD4 agonists and modulators, compounds targeted to HBcAg, compounds targeted to hepatitis B core antigen (HBcAg), covalently closed circular DNA (cccDNA) inhibitors, cyclophilin inhibitors, cytokines, cytotoxic T-lymphocyte-associated protein 4 (ipi 4) inhibitors, DNA polymerase inhibitors, endonuclease modulators, epigenetic modifiers, farnesol X receptor agonists, gene modifiers or editors, HBsAg inhibitors, HBsAg secretion or assembly inhibitors, HBV antibodies, HBV DNA replication inhibitors, HBV envelope protein inhibitors, HBV inhibitors, hbiap 1 inhibitors, HBV envelope protein inhibitors, HBV inhibitors, IL-2 agonists, IL-7 agonists, immunoglobulin G modulators, immunomodulators, indoleamine-2, ribonucleotide reductase inhibitors, interferon agonists, interferon alpha 1 ligands, interferon alpha 2 ligands, interferon alpha 5 ligand modulators, interferon alpha ligands, interferon alpha ligand modulators, interferon alpha receptor ligands, interferon beta ligands, interferon receptor modulators, interleukin-2 ligands, ipi4 inhibitors, lysine demethylase inhibitors, histone demethylase inhibitors, KDM5 inhibitors, KDM1 inhibitors, killer lectin-like receptor subfamily G member 1 inhibitors, lymphocyte activation gene 3 inhibitors, lymphotoxin beta receptor activators, microRNA (miRNA) gene therapy agents, axl modulators, B7-H3 modulators, B7-H4 modulators, CD160 modulators, CD161 modulators, CD27 modulators, CD47 modulators, CD70 modulators, GITR modulators, HEVEM modulators, ICOS modulators, mer modulators, NKG2A modulators, NKG2D modulators, OX40 modulators, SIRP α modulators, TIGIT modulators, tim-4 modulators, tyro modulators, na + -taurate cotransporter polypeptide (NTCP) inhibitors, natural killer cell receptor 2B4 inhibitors, NOD2 gene stimulators, nucleoprotein inhibitors, nucleoprotein modulators, PD-1 inhibitors, PD-L1 inhibitors, PEG-interferon λ, peptidyl prolyl isomerase inhibitors, phosphatidylinositol-3 kinase (PI 3K) inhibitors, recombinant Scavenger Receptor A (SRA) proteins, recombinant thymosin α -1, retinoic acid inducible gene 1 stimulators, reverse transcriptase inhibitors, ribonuclease inhibitors, RNADNA polymerase inhibitors, short interfering RNAs (sirnas), short synthetic hairpin RNAs (sshrnas)), SLC10A1 gene inhibitors, SMAC mimetics, src tyrosine kinase inhibitors, interferon gene Stimulators (STING), NOD1 stimulators, T cell surface glycoprotein CD28 inhibitors, T cell surface glycoprotein CD8 modulators, thymosin agonists, thymosin alpha 1 ligands, tim-3 inhibitors, TLR-3 agonists, TLR-7 agonists, TLR-9 agonists, TLR9 gene stimulators, toll-like receptor (TLR) modulators, viral ribonucleotide reductase inhibitors, zinc finger nucleases or synthetic nucleases (TALENs) and combinations thereof.

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 outcome 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.

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

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.

Pharmaceutical composition

In certain embodiments, lansoprazole, a derivative thereof, or a pharmaceutically acceptable salt thereof is administered in a pharmaceutical composition. 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 "handbook of pharmaceutical excipients" (1986). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextran, hydroxyalkyl cellulose, hydroxyalkyl methyl cellulose, stearic acid, and the like. The pH of the formulation ranges from about 3 to about 11, but is typically from about 7 to 10. In some embodiments, the pH of the formulation ranges from about 2 to about 5, but typically from about 3 to 4.

The formulations include those suitable for the aforementioned routes of administration. The formulations may conveniently be presented in unit dosage form and 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 invention 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.

Tablets are made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by: the active ingredient in free-flowing form, such as a powder or granules, is compressed in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersing agent. Molded tablets may be prepared by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and optionally formulated so as to provide sustained or controlled release of the active ingredient therefrom.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

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. The suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol, or as a lyophilized powder. Acceptable carriers and solvents that may be employed include water, ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Acceptable carriers and solvents that may be employed include water, ringer's solution, isotonic sodium chloride solution and hypertonic sodium chloride solution.

Additional objects, advantages and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples.

Examples

Example 1 evaluation of in vitro anti-HBV Activity of test Compound Lansoprazole Using HepG2-NTCP cells

The compound preparation method comprises the following steps:

in the case of preparation of a concentration of 10mM, the volume (. Mu.l) = sample mass (. Mu.l) × purity ÷ molecular weight ÷ 10X 10 of DMSO as solvent⁶

Control compounds include ETV (batch No.: P1214012;99.0% purity), available from Shanghai Tantake Technology, inc.; and RG7834 (RG 7834, also known as RO7020322, is a highly selective and orally bioavailable HBV inhibitor, effectively inhibits HBV antigens (HBsAg and HBeAg) and HBVDNA. Batch No.: ET25747-14-P1;99.5% purity), and is obtained from Shanghai drug Mingkuda New drug development, inc. The mother liquors of the above control compounds were all at 20mM concentration and stored at-20 ℃.

Table 1: primary reagents and cellular viruses

Experimental protocol

Plating cells and compound treatment

HepG2-NTCP planking

On day 0, hepG2-NTCP cells were seeded into 48-well cell plates (7.5X 104 cells/well).

Infectious virus and compound treatment

On day 2, cells were pretreated with the addition of compound for 2 hours, and then HepG2-NTCP cells were infected with the addition of D-type HBV (compound was added at the same time as infection). The tested compound is provided with 3 single-drug concentrations and 1 combined drug concentration, the three single-drug concentrations of the lansoprazole drug are 1, 10 and 20 mu M respectively, and the combined drug concentration is lansoprazole 20 mu M + ETV0.1nM; control compounds were at 7 concentrations of ETV and RG7834, respectively, with wells containing no compound in DMSO alone and a single drug concentration of ETV of 0.1nm,2 duplicate wells tested. 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 HBVDNA (qPCR), HBeAg and HBsAg (ELISA). After collecting the cell supernatant, cellTiter-Glo was added to detect cell viability, and the specific experimental procedure is shown in Table 3.

Table 2: concentration of the Compound

Table 3: experimental procedure

Sample detection

1) qPCR method for detecting HBVDNA content in cell culture supernatant

The DNA in the cell culture supernatant was extracted with reference to the QIAamp96DNABLOOD kit instructions. The sample volume was 120. Mu.l, and the DNA elution volume was 120. Mu.l of supernatant. qPCR was used to determine the HBVDNA content.

As shown in table 4, qPCR reaction mixtures were prepared.

TABLE 4.QPCR reaction Components Table

The qPCR reaction mix was added to 384 well reaction plates and 2 μ l of sample or standard was added to the corresponding wells, with a total volume of 10 μ l per reaction well. And (3) PCR reaction: 10 minutes at 95 ℃;95 ℃,15 seconds, 60 ℃,1 minute, 40 cycles.

2) ELISA method for detecting content of HBsAg and HBeAg in cell culture supernatant

The method refers to the kit specification, and the method is briefly described as follows: respectively taking 50 mu l of standard substance, adding the sample and the 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 by using washing liquid, then sucking to 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 luminescence value by using an enzyme-linked immunosorbent assay.

3) Cell viability assay

After the supernatant was collected, the medium and CellTiter-Glo were mixed in equal volumes, 50. Mu.l of each well was added to the cell plate, and the plate was shaken at room temperature in the dark for 10 minutes to measure the luminescence value.

4) Data computation

HBVDNA inhibition (%) = (1-number of copies of HBVDNA in compound group sample/number of copies of HBVDNA in DMSO group) × 100

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

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

Cytotoxicity% =100- [ (sample luminescence value-medium control luminescence value)/(DMSO control luminescence value-medium control luminescence value) ] × 100%

Data analysis

TABLE 5 percentage HBsAg and HBeAg inhibition for lansoprazole

TABLE 6 percentage of cytotoxicity (%) and percentage of HBVDNA inhibition (%) for lansoprazole

As shown in FIG. 1 and Table 5, table 6, 20. Mu.M and 10. Mu.M lansoprazole inhibited HBVDNA, HBsAg and HBeAg well in HepG2-NTCP cells, while 0.1nM ETV inhibited HBVDNA only well.

The test results show that compared with ETV, the lansoprazole drug group has obvious inhibition effects on HBVDNA, HBsAg and HBeAg, especially on HBsAg and HBeAg; lansoprazole has a remarkable HBV inhibiting effect, particularly an inhibiting effect on HBsAg and HBeAg, so that the lansoprazole can be used as a candidate drug for functionally curing hepatitis B and eliminating hepatitis B virus, and particularly when the lansoprazole is combined with a nucleoside analogue drug which can reduce the HBV titer but cannot reduce the HBsAg and HBeAg, for example, when the nucleoside analogue drug is used for reducing the virus titer, so that the virus content in a human body is reduced to a lower level, the lansoprazole is used for further eliminating the HBsAg and HBeAg, and further eliminating the virus, even completely curing the virus is expected.

While the invention has been described with reference to specific embodiments, those skilled in the art will recognize that changes or modifications can be made to the embodiments described without departing from the spirit and scope of the invention, which is defined by the claims.

Claims (11)

1. Use of compound 1, a derivative thereof or a pharmaceutically acceptable salt thereof, as sole active ingredient in the manufacture of a medicament for reducing the Hepatitis B Virus (HBV) load, HBsAg and/or HBeAg levels, wherein said derivative of compound 1 is selected from the deuterated products thereof,

2. the use of claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, malate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, thiocyanate, tosylate, undecanoate, sodium, calcium, potassium, ammonium, tetraethylammonium, methylammonium, dimethylammonium, and ethanolamine salts.

3. The use of claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of lansoprazole sulfonate and lansoprazole sodium salt.

4. The use of claim 1, wherein the derivative is selected from compound 1-2 to compound 1-4:

5. the use of claim 1, wherein the medicament is formulated for administration by a route selected from the group consisting of: oral, rectal, nasal, pulmonary, buccal and sublingual, vaginal, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural.

6. The use of claim 1, wherein the medicament is formulated for topical administration.

7. The use of claim 1, wherein the medicament is formulated for parenteral administration.

8. The use of claim 1, wherein the medicament is formulated for oral administration.

9. The use of claim 1, wherein the medicament is a tablet or capsule.

10. The use of claim 1, wherein the medicament is an enteric coated tablet.

11. Use of a pharmaceutical composition for the manufacture of a medicament for reducing the level of Hepatitis B Virus (HBV) load, HBsAg and/or HBeAg, wherein the pharmaceutical composition comprises a therapeutically effective amount of compound 1, a derivative thereof or a pharmaceutically acceptable salt thereof as the sole active ingredient, and a pharmaceutically acceptable carrier, said derivative being selected from the group consisting of compound 1-2 to compound 1-4:

Images