CN114259492A

CN114259492A - Application of nitazoxanide in treating hepatitis B

Info

Publication number: CN114259492A
Application number: CN202111572414.8A
Authority: CN
Inventors: 李瑛颖; 陈明键; 仇思念
Original assignee: China Israel Hyde Artificial Intelligence Drug Research And Development Co ltd
Current assignee: China Israel Hyde Artificial Intelligence Drug Research And Development Co ltd
Priority date: 2021-12-21
Filing date: 2021-12-21
Publication date: 2022-04-01

Abstract

The invention relates to an application of nitazoxanide in treating hepatitis B. In particular, the present invention relates to the use of nitazoxanide, alone or in combination with another therapeutic or prophylactic agent, for the treatment of hepatitis b and related pharmaceutical compositions.

Description

Application of nitazoxanide in treating hepatitis B

Technical Field

The invention belongs to the technical field of medicinal chemistry, and particularly relates to application of nitazoxanide in treating hepatitis B.

Background

Hepatitis B Virus (HBV) is the causative agent of hepatitis b (hepatitis b for short). HBV infection is a global public health problem, and persistent HBV infection can lead to cirrhosis and even liver cancer. Along with the production and investment of genetic engineering vaccines, the popularization rate of hepatitis B vaccines is increased year by year, and the infection rate of hepatitis B is in a descending trend. In China, because the cardinality of hepatitis B virus carriers is large, the prevention and treatment of hepatitis B is still the key point of public health in China at present and even in decades in the future.

Clinically, chronic hepatitis B patients are treated mainly by non-specific antiviral therapies, including the use of nucleoside analogs and type I interferons. 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 are effective in reducing HBV DNA levels and controlling hepatitis B virus levels in patients, they have no direct effect on the clearance of HBeAg and HBsAg due to their target of action being 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. The treatment course of the type I interferon is long, the cost is high, the lasting response rate of a patient is only 10-47%, and the treatment effect of the non-specific antiviral treatment on the hepatitis B patient is not ideal.

The hepatitis B vaccine is an effective means for preventing hepatitis B, and the conventional recombinant hepatitis B vaccine is mainly used for specifically preventing HBV infection of newborns and susceptible people. The immunization of the hepatitis B vaccine of the newborn effectively prevents new HBV infection. Wherein hepatitis B surface antigen (HBsAg) with natural structure is used as target antigen. However, this method can induce the production of antibodies only in uninfected healthy humans, and thus exerts a prophylactic effect, and is a prophylactic vaccine.

The persistent state of chronic HBV infection is mainly due to the immune tolerance of the body to the hepatitis b virus antigen. The immunoprophylactic hepatitis B vaccine is not effective to patients already suffering from chronic HBV infection, and has no treatment effect. If patients with chronic HBV infection are not treated, 25-40% of infected patients will progress to decompensated cirrhosis or liver cancer. Therefore, many scholars all over the world try to eliminate the therapeutic vaccine of chronic HBV infected liver cells by inducing chronic HBV infected persons to generate specific immune response reaction to HBV viral antigen and mobilizing the specific immunity of the organism, and the existing therapeutic hepatitis B vaccine has certain advantages but also has some problems. For example, DNA vaccines induce strong cellular and humoral immune responses and break specific Cytotoxic T Lymphocyte (CTL) tolerance, but the problems of safety (including environmental safety and human safety) and standardization remain to be studied intensively. Polyepitope gene vaccines or polyepitope polypeptide vaccines provide a new idea for multivalent vaccine research, but are limited to patients expressing certain MHC molecules due to the limitations of MHC molecules, and there is also a need to overcome the inherent disadvantages of poor immunogenicity of epitope vaccines. The immunogenicity of the antigen-antibody complex therapeutic vaccines is also limited by the use of conventional aluminum salt adjuvants.

Antiviral treatments are currently marketed mainly by using interferons or nucleoside analogues. Wherein the nucleoside analog inhibits HBV production by inhibiting reverse transcriptase activity during HBV replication. Although the reverse transcriptase inhibitor can control the level of hepatitis B virus of a patient, the reverse transcriptase inhibitor is high in cost and lacks targeting property, most of the reverse transcriptase inhibitor is discharged through the kidney after being taken, only a few parts of the reverse transcriptase inhibitor are absorbed by liver organs, and the treatment effect is poor.

Disclosure of Invention

In order to improve the technical problems, the invention provides the application of Nitazoxanide (NTZ) or pharmaceutically acceptable salts or analogues thereof in preparing a medicament for treating or preventing viral hepatitis. The invention also provides the application of the combination of nitazoxanide or pharmaceutically acceptable salt or analogue thereof and another therapeutic agent or prophylactic agent in the preparation of medicaments for treating or preventing viral hepatitis. Preferably, the use comprises reducing the Hepatitis B Virus (HBV) load and/or HBsAg (hepatitis b surface antigen) level in an individual in need thereof.

The inventors have surprisingly found that the drug nitazoxanide, which is commonly used for killing and inhibiting parasites, has the ability to reduce the Hepatitis B Virus (HBV) load and/or the level of HBsAg (hepatitis b surface antigen) in cell tests and animal tests, and thus is expected to achieve the effect of eliminating hepatitis b virus, thereby achieving a complete cure of hepatitis b.

According to an embodiment of the invention, the viral hepatitis comprises hepatitis b or hepatitis d.

According to the invention, the medicament is particularly capable of reducing the Hepatitis B Virus (HBV) load and/or HBsAg levels.

Accordingly, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of nitazoxanide or a pharmaceutically acceptable salt or analog thereof and optionally an additional therapeutic or prophylactic agent.

According to an embodiment of the invention, nitazoxanide or a pharmaceutically acceptable salt or analogue thereof enhances the therapeutic and/or prophylactic effect of the optional further therapeutic or prophylactic agent on viral hepatitis. Thus, the subject matter of the present invention also relates to the use of nitazoxanide or a pharmaceutically acceptable salt or analogue thereof for the potentiation of the treatment and/or prevention of viral hepatitis optionally with a further therapeutic or prophylactic agent.

According to an embodiment of the invention, the additional therapeutic or prophylactic agent is an antiviral drug, preferably an anti-hepatitis b virus drug, including interferon or PEG-interferon and nucleoside analogues, more preferably the second therapeutic or prophylactic agent may be selected from lamivudine, adefovir, entecavir, tenofovir or derivatives thereof, such as tenofovir disoproxil fumarate, tenofovir alafenamide and the like.

According to an embodiment of the invention, the pharmaceutical composition is formulated for administration by a route selected from the group consisting of: oral, injection, rectal, nasal, pulmonary, topical, buccal and sublingual, vaginal, parenteral, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural.

According to an embodiment of the invention, the pharmaceutical composition is preferably administered orally.

The oral dosage form is not particularly limited, and any oral dosage form known in the art may be used, and preferably, oral dosage forms known in the art including tablets, capsules, suspensions, or oral solutions are included. For oral administration, the dosage standard is, for example, 500-1500 mg/day of nitazoxanide or a pharmaceutically acceptable salt or an analog thereof, preferably 700-1200 mg/day, preferably 800-1000 mg/day, and most preferably 1000 mg/day of nitazoxanide or a pharmaceutically acceptable salt or an analog thereof.

The time of administration of the pharmaceutical composition according to the invention may depend on the extent of the disease, preferably at least 1 month, for example, it may be 1, 2, 3, 4, 5 or 6 months, and may be up to lifelong due to the needs of the disease.

According to an embodiment of the invention, the ratio of the amount of nitazoxanide to the additional therapeutic or prophylactic agent in the medicament or pharmaceutical composition according to the invention is preferably 100:1 to 10:1, preferably 50:1 to 20:1, more preferably 30:1 to 25:1, by weight. According to the present invention, it was confirmed that the amount of nitazoxanide in this range and the additional therapeutic agent or prophylactic agent can produce a synergistic effect therebetween, increasing the HBV DNA inhibitory rate.

According to an embodiment of the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient selected from at least one of the following excipients, including but not limited to: fillers, disintegrants, binders, lubricants, surfactants, flavoring agents, wetting agents, pH adjusting agents, solubilizers or cosolvents, and osmotic pressure adjusting agents. The skilled person can easily determine how to select the corresponding excipients and the corresponding amounts thereof according to the needs of a particular dosage form.

Terms and explanations

As used herein, the term "nitazoxanide" refers to [2- [ (5-nitro-1, 3-thiazol-2-yl) carbamoyl ] phenyl ] acetate (CAS registry number 55981-09-4) having the following structure:

nitazoxanide is a derivative of nitrosalicylic acid amide, the actual mechanism of action of which is not yet clarified, but is thought to be involved in the inhibition of the enzyme-dependent electron transfer reaction of pyruvate, ferredoxin oxidoreductase, the latter being important for anaerobic energy metabolism. The product is also active against various intestinal parasites such as sporozoite of Behcet, amoeba, ascaris lumbricoides, hookworm, trichuris, beef tapeworm, short-shell tapeworm and fasciola hepatica, in addition to cryptosporidium and giardia intestinalis. The use of derivatives of nitrosalicylic acid amide in the treatment or prevention of viral hepatitis is currently under investigation, and nitazoxanide has been approved by the U.S. FDA for use in the treatment of various viral and parasitic infections.

Herein, the term "pharmaceutically acceptable salt" refers to a salt made from a pharmaceutically acceptable non-toxic acid (including inorganic or organic acids). The compounds of the invention may be mono-, di-or tri-salts depending on the number of acidic functional groups present in the free base form of the compound. By way of example, the acids include acetic acid, trifluoroacetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucose, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, oxalic acid, tartaric acid, p-toluenesulfonic acid and the like.

Preferably, the nitazoxanide or a pharmaceutically acceptable salt thereof is, for example, a hydrochloride.

Preferably, the analogues of nitazoxanide include Tizoxanide (Tizoxanide) and deuterated nitazoxanide. In addition, tizoxanide is generally considered an active metabolite of nitazoxanide, having the following structure:

examples of deuterated nitazoxanide can include, but are not limited to, the following and deuterated protocol combinations thereof:

correspondingly, deuterated forms of tizoxanide include the structures shown below:

as used herein, "an individual in need thereof" includes individuals infected with/carrying hepatitis B virus or hepatitis D virus, particularly patients suffering from acute, chronic, severe hepatitis or post-hepatitis cirrhosis, carriers of hepatitis B surface antigen, and patients infected with hepatitis D, and the like.

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 (HBV), a DNA virus whose genome is a double-stranded, circular, incompletely closed DNA. The outermost layer of the virus is the outer membrane or coat of the virus, the inner layer is the core, and the nucleoprotein is the core antigen (HBcAg) and cannot be detected in 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.

Marker detection of hepatitis b is as follows: (ii) 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. (vii) 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, the term "additional therapeutic or prophylactic agent" generally refers toA therapeutic or prophylactic agent selected from an interferon or a nucleoside analogue. In a preferred embodiment, the nucleoside analogue is selected from one or a combination of two or more, preferably a combination of at least two, of entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide.

Tenofovir disoproxil fumarate (the name of England: (TDF); (R) - [ [2- (6-amino-9H-purin-9-yl) -1-methylethoxy ] methyl ] phosphonic acid diisopropoxycarbonylmethyl 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 Gilidard company in the United states following Adefovir dipivoxil, is first marketed in the United states in 10 months in 2001, and is currently marketed in countries such as Europe, Australia, and Canada.

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 orally taken, the tenofovir is phosphorylated by cell kinase to generate a metabolite tenofovir diphosphate with pharmacological activity, the tenofovir diphosphate competes with 5 '-triphosphate deoxyadenosine monophosphate 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 (TAF), 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, the tenofovir alafenamide only needs one tenth of TDF administration dosage to achieve the same antiviral curative effect 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.

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 synthesis of entecavir is disclosed in WO 9809964; WO0164421 discloses low dose entecavir solid formulations.

Entecavir is a highly effective antiviral agent, developed by schrobo corporation in the 90 s of the 20 th century, and has a strong anti-HBV effect. It can be phosphorylated to active triphosphate, which has a half-life in cells of 15 h. 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 start of HBV polymerase; (2) formation of a reverse transcribed negative strand of a pregenomic mRNA; (3) synthesis of HBV DNA plus strand.

However, these nucleoside analogs have the defect of not clearing hepatitis B virus, can only block virus replication by taking a long-term medicine, and have no clearing effect on HBsAg and HBeAg.

As used herein, a "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 the desired therapeutic result. The therapeutically effective amount of the pharmaceutical composition will depend on the nature of the disease or condition and the particular drug, and can be determined by standard clinical techniques known to those skilled in the art.

Treatment outcomes may include alleviation of symptoms, prolongation of survival, increased mobility, and the like. The therapeutic result is not necessarily a "(complete) cure". The therapeutic outcome may also be prophylactic.

The medicament or pharmaceutical composition of the present application is administered by any route suitable for the disease 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. It will be appreciated that the preferred route may vary depending on, for example, the condition of the recipient. One advantage of the medicaments or pharmaceutical compositions according to the invention is that they are orally bioavailable and can be administered orally.

In a preferred embodiment, the pharmaceutical 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.

The pharmaceutical compositions according to the invention 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). 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 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.

Tablets are made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by: the active ingredient in a 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 dispersant. 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 from the tablet.

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.

Advantageous effects

According to the invention, nitazoxanide has obvious inhibiting effect on hepatitis B surface antigen (HBsAg), and in cell and animal experiments, nitazoxanide can obviously reduce HBV DNA content and HBsAg level. In addition, if nitazoxanide is combined with the existing nucleoside analogue, the combination has synergistic effect in reducing the HBV DNA content and the HBsAg level in an individual, and the HBsAg is expected to turn negative. Nitazoxanide can be used alone or optionally in combination with another therapeutic/prophylactic agent for preparing antiviral drugs, especially anti-hepatitis B virus drugs, thereby providing a novel means for preventing and treating viral hepatitis, not only reducing HBV DNA content and HBsAg level, but also hopefully eliminating hepatitis B virus from individuals.

Drawings

FIG. 1 is a graph showing the HBsAg inhibition rate of HepG2.2.15 cells of example 1 after the action with nitazoxanide;

FIG. 2 is a graph of cytotoxicity of HepG2.2.15 cells of example 1 after exposure to nitazoxanide;

FIG. 3 is a graph showing the HBV DNA content in the plasma of mice under different action times of the excipient and different drugs in example 3;

FIG. 4 is a graph showing the HBV DNA content in the plasma of mice under different action times of the different drugs in example 4;

FIG. 5 is a graph showing the synergistic effect of nitazoxanide and tenofovir alafenamide in example 2;

figure 6 is a summary of the effect of nitazoxanide and Tenofovir Alafenamide (TAF), alone or in combination, in example 2, wherein HD117 corresponds to nitazoxanide, in inhibiting HBsAg at different concentrations.

Detailed Description

Hereinafter, the use of nitazoxanide in hepatitis B will be described in more detail with reference to specific examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

Example 1:

the inhibitory effect of nitazoxanide on HBV was determined according to the test procedure shown below.

Compound (I)

Nitazoxanide (NTZ) and Tenofovir Alafenamide (TAF) were purchased from shanghai ceramic biotechnology limited. Entecavir (ETV) available from shanghai tanacetaceae technologies ltd (batch No.: P1214012; 99.0% purity).

Taking the preparation of 20mM concentration as an example, the compound stock solution is prepared as follows, and is stored at-20 ℃ after the preparation is completed:

volume of solvent DMSO (μ l) ═ sample mass (mg) × purity ÷ molecular weight ÷ 20 × 10 ÷ molecular weight ÷ sample mass⁶。

Culture medium

Culture medium for frozen PHH culture: mainly DMEM medium (Gibco cat # 11960051) containing 10% fetal bovine serum (FBS, hyclone cat # SV3008703) and 1% penicillin-streptomycin was used mainly for cell culture.

Culture medium for frozen PHH plating: mainly contains 10% of fetal bovine serum (FBS, Hyclon cat # SV3008703) 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.

Other test materials

Table 1: other reagents and cells

Example 1 cytological Studies of Nitazoxanide with Entecavir

Test method

A. Cell culture, cell plating and compound treatment

On day 0, HepG2.2.15 cells (hepatitis B virus-stabilized cells) were plated in 96-well plates (cell density 6X 10)⁴Individual cells/well).

On day 1, cells were added to compound-containing medium at 9 concentrations of test compound, 3-fold dilution, triplicate wells at 50 μ M, 16.6667 μ M, 5.5556 μ M, 1.8519 μ M, 0.6173 μ M, 0.2058 μ M, 0.0686 μ M, 0.0229 μ M, 0.0076nM, 9 concentrations of the control compound Entecavir (ETV), 3-fold dilution, triplicate wells at 20nM, 6.6667nM, 2.2222nM, 0.7407nM, 0.2469nM, 0.0823nM, 0.0274nM, 0.0091nM, 0.003 nM. Cells were placed in 5% CO₂And cultured at 37 ℃ for 3 days.

On day 4, the fresh medium containing the compound was changed once (compound concentration was unchanged) and the cells were placed in 5% CO₂And cultured at 37 ℃ for 3 days.

On day 7, cell culture supernatants were collected and tested for HBsAg by ELISA and HBV DNA levels by qPCR. Meanwhile, CellTiter-Glo measures cell viability (see section B below in this example for details of the measurement method); cells were collected and stored frozen.

The compound test results are shown in Table 2 below

TABLE 2

B. Sample detection

The qPCR method is adopted to detect the content of HBV DNA in the cell culture supernatant:

DNA was extracted from cell culture supernatants with reference to QIAamp 96DNA kit instructions. The content of HBV DNA was determined by qPCR. And (3) PCR reaction: at 95 ℃ for 10 min; 95 ℃, 15sec, 60 ℃, 1min, 40 cycles.

The ELISA method is used for detecting the content of HBsAg in cell culture supernatant:

the detection method is carried out according to the instruction of the kit, 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 1:1 dilution) was added to each well, incubated at room temperature for 10 minutes, and the luminescence was measured with a microplate reader.

C. Data analysis

HBV DNA inhibition (%) (HBV copy number of 1-compound group sample/HBV copy number of DMSO group) × 100%;

HBsAg inhibition (%) is ═ 100% (1-HBsAg value of sample/DMSO control HBsAg value);

cytotoxicity (%) is 1- ((value of sample of compound group-average of blank group)/(average of DMSO group-average of blank group) × 100%;

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

The test results are shown in FIGS. 1 and 2. It can be seen that nitazoxanide has a significant inhibitory effect on HBsAg in cells at least at concentrations of 1.8519 μ M and 0.6173 μ M, and that the cytotoxicity of nitazoxanide is stable.

Example 2 cytological Studies of Nitazoxanide and Tenofovir alafenamide

In this example, the assay procedure involved essentially the same procedure as in example 1, except that the concentrations were tested using the compounds of Table 3 below and the HepG2.2.15 cells were replaced with HepG2-NTCP cells.

TABLE 3 test concentrations of the respective Compounds

Index of test acceptance

With ETV as a control, if the control compound in the assay gave the expected result, the other data in the assay were deemed valid and the data were analyzed.

Analysis of results

Referring to fig. 5, fig. 6 and table 4, it can be seen that nitazoxanide, when used in combination with Tenofovir Alafenamide (TAF), has a synergistic/additive effect in inhibiting HBsAg.

Specifically, to more clearly analyze the synergistic effect of nitazoxanide and tenofovir alafenamide, a Combination Index analysis (CI) software CompuSyn software V1.0 software was used, and the analysis was performed by the Non-Constant Combo method, and the results are shown in Table 4.

In contrast, results for Entecavir (ETV) are shown in table 5.

TABLE 4 analysis of the combined effect of nitazoxanide + TAF on HBsAg inhibition

Data points	Nitazoxanide concentration (μ M)	TAF concentration (nM)	Inhibition ratio (%)	CI value	Description of the results
						1	2.0	50.0	92.17	0.8571	Synergistic effect
2	0.5	50.0	40.09	0.8390	Synergistic effect
						3	0.2	50.0	20.71	0.6533	Synergistic effect
4	2.0	15.0	90.80	0.9317	Additive effect
						5	0.5	15.0	37.50	0.8795	Synergistic effect
6	2.0	5.0	90.35	0.9552	Additive effect
						7	0.5	5.0	39.46	0.8438	Synergistic effect
8	0.2	5.0	21.82	0.5146	Synergistic effect

TABLE 5ETV test results

The above test results show that, first, the test results of the control compound ETV are in line with expectations, and therefore the overall test results fall within acceptable ranges.

Secondly, the combined medication test and the statistical analysis of the nitazoxanide and the TAF show that the combination of the nitazoxanide and the TAF can effectively reduce the HBsAg and has obvious synergistic action or addition action.

Particularly, the combination of the two has obvious synergistic effect when the concentration of the TAF is 50nM and the concentration of the nitazoxanide is 0.2-2.0 muM, or the combination of the two has obvious synergistic effect when the concentration of the TAF is 5nM and the concentration of the nitazoxanide is 0.2-0.5 muM and the concentration of the TAF is 15nM and the concentration of the nitazoxanide is 0.5 muM.

According to the results, the combination of the nifedipine with the effective treatment amount and the tenofovir alafenamide with the effective treatment amount can generate addition action and synergistic action, and is expected to become a new medicine treatment combination for hepatitis B.

Example 3 zoological Studies of Nitazoxanide and Tenofovir alafenamide

6-8 week old C57BL/6 male mice were used in the AAVHBV mouse model.

One recombinant virus carries 1.3 copies of the HBV genome (genotype D) and is packaged in the AAV serotype 8(AAV8) capsid.

Adult C57BL/6 mice were injected via tail vein injection with the indicated amount of recombinant virus (diluted in phosphate buffered saline) and after 6-8 weeks, the mice were divided into two groups and received different test sample doses. Levels of HBV surface antigen (HBsAg) and HBV DNA in serum were monitored weekly.

TABLE 6 test protocol

Grouping	Medicine	Mouse administration dose	Mode of administration
				G1	Excipient	0 (physiological saline)	Orally administered once a day
G2	Tenofovir Alafenamide (TAF)	3.75mpk	Orally administered once a day
				G6	Nitazoxanide (NTZ)	100mpk	Orally administered once a day
G8	NTZ+TAF	100 mpk/day +3.75 mpk/day	Orally administered once a day

The test result is shown in figure 3, although the inhibition effect of orally administered nitazoxanide on HBV DNA is not obvious, the combination of the nitazoxanide and tenofovir alafenamide has obvious synergistic effect and can enhance the inhibition on the HBV DNA.

EXAMPLE 4 zoology study of Nitazoxanide

And (3) an in vivo inhibition test of single intraperitoneal administration of nitazoxanide on HBV (hepatitis B Virus) of AAVHBV mice.

TABLE 7 test protocol

The results of the experiment are shown in fig. 4, although the reduction of HBV DNA by intravenous administration of nitazoxanide alone was not significant as demonstrated in example 3, there was a significant reduction of HBV DNA of about 1log by changing the route of administration, i.e. by intraperitoneal administration.

Therefore, in cell experiments and animal experiments, nitazoxanide respectively shows strong inhibition effect on HBsAg in vitro and potential HBV DNA inhibition in vivo, although the action mechanism of the nitazoxanide in vivo and in vitro experiments is not necessarily completely the same, the tests show that the nitazoxanide is a potential drug with prospect for treating hepatitis B. Also, in case of combining nitazoxanide with another therapeutic/prophylactic agent such as tenofovir alafenamide, a synergistic effect can be achieved, thereby enhancing the inhibition of HBV DNA.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of nitazoxanide or a pharmaceutically acceptable salt or analogue thereof, alone or in combination with another therapeutic or prophylactic agent, in the manufacture of a medicament for the treatment or prevention of viral hepatitis.

2. The use of claim 1, wherein the viral hepatitis comprises hepatitis b or hepatitis delta.

3. The use of claim 1 or 2, wherein the medicament reduces Hepatitis B Virus (HBV) load and/or HBsAg levels.

4. The use according to any one of claims 1 to 3, wherein the additional therapeutic or prophylactic agent is an antiviral drug, preferably an anti-hepatitis B virus drug, comprising interferon or PEG-interferon and nucleoside analogues; more preferably, the additional therapeutic or prophylactic agent is selected from lamivudine, adefovir, entecavir, tenofovir or derivatives thereof, such as tenofovir disoproxil fumarate, tenofovir alafenamide.

5. A pharmaceutical composition comprising a therapeutically effective amount of nitazoxanide or a pharmaceutically acceptable salt or analog thereof and optionally an additional therapeutic or prophylactic agent.

6. The pharmaceutical composition of claim 5, which is an oral dosage form.

7. The pharmaceutical composition according to claim 5 or 6, wherein the additional therapeutic or prophylactic agent is an antiviral drug, preferably an anti-hepatitis B virus drug, comprising interferon or PEG-interferon and nucleoside analogues; more preferably, the additional therapeutic or prophylactic agent is selected from lamivudine, adefovir, entecavir, tenofovir or derivatives thereof, such as tenofovir disoproxil fumarate, tenofovir alafenamide.

8. The pharmaceutical composition of claim 6 or 7, wherein the oral dosage form comprises a tablet, capsule, suspension, or oral solution; for oral administration, the dosage standard is, for example, 500-1500 mg/day of nitazoxanide or a pharmaceutically acceptable salt or an analog thereof, preferably 700-1200 mg/day, preferably 800-1000 mg/day, and most preferably 1000 mg/day of nitazoxanide or a pharmaceutically acceptable salt or an analog thereof.

9. The pharmaceutical composition according to any one of claims 6 to 8, wherein the ratio of the amount of nitazoxanide to the additional therapeutic or prophylactic agent is preferably 100:1 to 10:1, preferably 50:1 to 20:1, more preferably 30:1 to 25:1, by weight.

10. The pharmaceutical composition according to any one of claims 6 to 9, further comprising a pharmaceutically acceptable excipient selected from at least one of the following excipients: fillers, disintegrants, binders, lubricants, surfactants, flavoring agents, wetting agents, pH adjusting agents, solubilizers or cosolvents, and osmotic pressure adjusting agents.