CN114681439A - Application of fenretinide in preparation of medicine for treating or preventing viral hepatitis - Google Patents

Abstract

The invention provides an application of fenretinide 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 fenretinide in preparation of medicine for treating or preventing viral hepatitis

Technical Field

The invention relates to the technical field of antiviral drugs, in particular 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. China is a big country with hepatitis B, and hepatitis B virus carriers are close to 1.3 hundred million people, accounting for about 9 percent of the total population. Although the new hepatitis B infection rate is effectively controlled along with the wide popularization of hepatitis B vaccines, the population base of hepatitis B carrying population is large, and the prevention and treatment of hepatitis B become the most important public health problem in China. 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 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 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 inhibitors can control the level of hepatitis B virus in patients, the problems of drug resistance, high medical cost, serious side effects of the drugs 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.

Fenretinide (Fenretinide) is a vitamin A analogue, can bind to Retinoic Acid Receptor (RAR) to induce cell death, and can inhibit the growth of various tumor cells, including small cell lung cancer cells, malignant hematopoietic cells and breast cancer cells. Fenretinide also protects women against ovarian cancer development.

At present, the researches aiming at the indication of fenretinide mainly aim at the application of the fenretinide in treating or preventing diseases such as tumor and the like, and no research report is available for treating or preventing viral hepatitis.

Disclosure of Invention

The present invention provides a novel viral hepatitis treatment option by applying compound 1, fenretinide, its derivatives or pharmaceutically acceptable salts thereof, for the treatment or prevention of viral hepatitis.

In one aspect, the invention provides the use of fenretinide 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, toluenesulfonate, undecanoate, sodium, calcium, potassium, ammonium, tetraethylammonium, methylammonium, dimethylammonium and ethanolamine salts, preferably fenretinide glucuronide monosodium 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.

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, nitazoxanide or an analog thereof, a compound of formula A, or a nucleoside analog,

formula A

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.

In another aspect, the present invention provides a pharmaceutical composition for treating or preventing viral hepatitis comprising a therapeutically effective amount of fenretinide 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. fenretinide or a pharmaceutically acceptable salt thereof is applied to treating or preventing viral hepatitis, thereby providing a novel viral hepatitis treatment option.

2. Fenretinide or pharmaceutically acceptable salt thereof can effectively reduce the load of Hepatitis B Virus (HBV), the HBsAg level and/or the HBeAg level, has 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. Fenretinide or pharmaceutically acceptable salt thereof has excellent clinical safety and pharmacokinetic property, and has better druggability.

4. Fenretinide or a pharmaceutically acceptable salt thereof can optionally be combined with one or more additional therapeutic or prophylactic agents, in particular with drugs that lower viral titers but do not completely eliminate viruses and do not lower HBsAg and/or HBeAg levels, eliminate hepatitis b virus from different aspects with the possibility of synergy.

Description of the drawings:

FIG. 1: the combined action of different concentrations of the fenretinide, 0.1nM of the entecavir, 10 mu M of the fenretinide and 0.1nM of the entecavir can inhibit the Hepatitis B Virus (HBV) load.

FIG. 2: the inhibition effect of the Fengwei A amine, 0.1nM entecavir, 10 mu M Fengwei A amine and 0.1nM entecavir on HBsAg is realized.

FIG. 3: the inhibition effect on HBeAg is realized by the combined action of fenretinide with different concentrations, 0.1nM entecavir, 10 mu M fenretinide and 0.1nM entecavir.

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 (namely fenretinide) with the hepatitis B treatment effect is screened out, and further through verification of biological experiments, the compound with the general formula 1 with the effect of removing HBsAg and HBeAg is obtained, so that the functional cure of hepatitis B and the removal of hepatitis B virus are expected.

In one aspect, the present invention provides the use of fenretinide, a derivative thereof 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 derivative of fenretinide comprises deuterated fenretinide, such as compound 2, compound 3, or fenretinide deuterated at other sites, such as methyl, benzene rings, and the like. For those skilled in the art, deuterated compounds do not change the original properties of the compound, but can slow the metabolic process, thereby extending the half-life. Can more effectively play the role of the medicine.

In a preferred embodiment, the pharmaceutically acceptable salt is fenretinide monosodium 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.

Fenretinide (Fenretinide) is a vitamin A analog, can bind to Retinoic Acid Receptor (RAR) to induce cell death, and can inhibit the growth of various tumor cells, including small cell lung cancer cells, malignant hematopoietic cells and breast cancer cells. Fenretinide also protects women against ovarian cancer development. 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 as 3 particles, 22nm in diameter, circular and filamentous, and less 42 nm in diameter. The spherical particles of (a), also known as Dane's particles, are intact HBV particles.

The markers for hepatitis b were detected 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.

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 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 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.

Tenofovir disoproxil fumarate (British name: Tenofovir disoproxil fumarate, TDF; chemical name is (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 (Tenofovir Alafenamide), 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.

In addition to the above active agents, the medicaments or pharmaceutical compositions described herein may optionally 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 (ccc) inhibitors, cyclophilin inhibitors, cytokines, cytotoxic T-lymphocyte-associated protein 4(ipi4) inhibitors, DNA polymerase inhibitors, endonuclease modulators, epigenetic modifiers, farnesoid X receptor agonists, gene modifiers or editors, HBsAg inhibitors, HBsAg secretion or assembly inhibitors, HBV antibodies, HBV DNA polymerase inhibitors, HBV replication inhibitors, HBV rnase inhibitors, HBV vaccines, HBV viral entry inhibitors, HBx inhibitors, hepatitis b large envelope protein modulators, hepatitis b large envelope protein stimulators, hepatitis b structural protein modulators, hepatitis b surface antigen (HBsAg) inhibitors, hepatitis b surface antigen (HBsAg) secretion or assembly inhibitors, hepatitis b virus E antigen inhibitors, hepatitis b virus replication inhibitors, hepatitis virus structural protein inhibitors, HIV-1 reverse transcriptase inhibitors, hyaluronidase inhibitors, IAP 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, 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, SIRPa modulators, TIGIT modulators, Tim-4 modulators, Tyro modulators, Na + -taurate cotransporter (NTCP) inhibitors, natural killer cell receptor 2B4 inhibitors, NOD2 gene stimulators, nucleoprotein inhibitors, nucleoprotein modulators, PD-1 inhibitors, PD-L1 inhibitors, PEG-interferon lambda, peptidyl prolyl isomerase inhibitors, phosphatidylinositol-3 kinase (PI3K) inhibitors, recombinant Scavenger Receptor A (SRA) proteins, recombinant thymosin alpha-1, retinoic acid-inducible gene 1 stimulators, reverse transcriptase inhibitors, ribonuclease inhibitors, RNA polymerase inhibitors, short interfering RNA (siRNA), short synthetic hairpin RNA (sshRNA), SLC10a1 gene inhibitors, SMAC mimetics, Src tyrosine kinase inhibitors, interferon gene Stimulator (STING) agonists, 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, a 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, fenretinide, 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 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 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 of ordinary skill in the art upon examination of the following examples.

Examples

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

The compound preparation method comprises the following steps:

in the case of preparation of a concentration of 10mM, the volume (μ l) of the solvent DMSO is the mass of the sample (mg) x purity ÷ molecular weight ÷ 10 × 10 ÷ molecular weight⁶

Control compounds include ETV (batch No.: P1214012; 99.0% purity), available from Shanghai Tantake Technology, Inc.; and RG7834(RG7834, also known as RO 7020322, is a highly selective and orally bioavailable HBV inhibitor, effectively inhibits HBV antigens (HBsAg and HBeAg) and HBV DNA. batch No.: ET 25747-14-P1; 99.5% purity), and is obtained from Shanghai drug, Congde, 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 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). The tested compound is provided with 3 single-drug concentrations and 1 combined drug concentration, the three single-drug concentrations of the fenretinide drug are respectively 0.1, 1 and 10 mu M, and the combined drug concentration is fenretinide 10 mu M + ETV 0.1 nM; control compounds were at 7 concentrations of ETV and RG7834 each, with wells containing only DMSO and no compound set up and ETV single drug concentration 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 HBV DNA (qPCR), HBeAg and hbsag (elisa). After collecting the cell supernatant, CellTiter-Glo was added to test the cell viability, and the specific procedure of the experiment is shown in Table 3.

Table 2: concentration of the Compound

Table 3: experimental procedure

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 sample volume was 120. mu.l, and the DNA elution volume was 120. mu.l of supernatant. qPCR detects the HBV DNA content.

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

TABLE 4.qPCR reaction composition Table

PCR reaction solution Components	1 volume required for the reaction system
		FastStart Universal Probe Master(2×)	5μl
Forward primer (10. mu.M)	0.4μl
		Reverse primer (10. mu.M)	0.4μl
Probe (10 μ M)	0.2μl
		AE	2μl

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, sample and reference substance, adding 50 mu l of enzyme conjugate into each hole, incubating for 60 minutes at 37 ℃, washing the plate with washing liquor, sucking dry, 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.

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

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

HBsAg inhibition (%) was ═ 100 (1-HBsAg value of sample/DMSO control HBsAg value) × 100

HBeAg inhibition (%) was (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

As shown in figure 1, the inhibition rate of 10 μ M fenretinide on HBV DNA in HepG2-NTCP cells reaches 65.54%, while 1 μ M fenretinide and 0.1 μ M fenretinide have no obvious inhibition effect on HBV DNA; the inhibition rate of 0.1nM ETV on HBV DNA reaches 57.28%; the inhibition rate of 10 mu M fenretinide +0.1nM ETV on HBV DNA reaches 66.38%.

The inhibition rate of the 10 mu M fenretinide on HBV DNA is basically the same as that of ETV of 0.1nM, and when the 10 mu M fenretinide is used in combination with the ETV of 0.1nM, the inhibition rate on the HBV DNA is not obviously improved compared with that of the 10 mu M fenretinide.

As shown in figure 2, the inhibition rate of 10 μ M fenretinide on HBsAg in HepG2-NTCP cells reaches 97.78%; 1 mu M, 0.1 mu M fenretinide and 0.1nM ETV have no obvious inhibition effect on HBsAg; the inhibition rate of 10 mu M fenretinide and 0.1nM ETV on HBsAg reaches 98.36%.

Compared with the situation that ETV of 0.1nM has no obvious inhibition effect on HBsAg, the inhibition effect of fenretinide of 10 mu M on HBsAg is obvious, and the inhibition effect of fenretinide of high dose on HBsAg is better than that of low dose group; when 10 mu M fenretinide is used together with 0.1nM ETV, the inhibition rate of HBsAg is not obviously improved compared with 10 mu M fenretinide.

As shown in FIG. 3, the inhibition rate of 10 μ M fenretinide on HBeAg in HepG2-NTCP cells reaches 95.57%, while 1 μ M fenretinide and 0.1 μ M fenretinide have no obvious inhibition effect on HBeAg; ETV of 0.1nM has no obvious inhibitory effect on HBeAg; the inhibition rate of 10 mu M fenretinide and 0.1nM ETV on HBeAg reaches 95.33%.

Compared with the ETV of 0.1nM without obvious inhibition effect on HBeAg, the fenretinide with high dosage can better play the inhibition effect on HBeAg; when the 10 mu M fenretinide is used together with 0.1nM ETV, the inhibition rate of HBeAg is not obviously improved compared with that of 10 mu M fenretinide.

The test results show that compared with ETV, the fenretinide 10 mu M drug group has obvious inhibition effect on HBV DNA, HBsAg and HBeAg, and especially achieves the inhibition effect of more than 95% on HBsAg and HBeAg; fenretinide has obvious HBV inhibiting effect, especially the inhibiting effect on HBsAg and HBeAg, so that fenretinide can be used as a candidate medicine for functionally curing hepatitis B and eliminating hepatitis B virus, especially when being combined with nucleoside analogue medicines which can reduce HBV titer but can not reduce HBsAg and HBeAg, for example, when nucleoside analogue medicines are used for reducing the titer of virus firstly, and the virus content in human body is reduced to a lower level, fenretinide is used for further eliminating HBsAg and HBeAg, thereby being expected to further eliminate virus even completely cure the virus.

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 described embodiments without departing from the spirit and scope of the invention, which is defined by the appended claims.

Claims (10)

1. The use of compound 1, a derivative thereof or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of viral hepatitis,

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, toluenesulfonate, undecanoate, sodium, calcium, potassium, ammonium, tetraethylammonium, methylammonium, dimethylammonium and ethanolamine salts, preferably fenretinide glucuronide monosodium salt.

3. The use of any one of claims 1-2, wherein the viral hepatitis is hepatitis b.

4. The use of claim 3, wherein the medicament is for reducing Hepatitis B Virus (HBV) load, HBsAg and/or HBeAg levels.

5. The use of any one of claims 1-4, wherein the medicament further comprises one or more additional therapeutic or prophylactic agents, wherein the additional therapeutic or prophylactic agent is selected from at least one of an interferon, a PEGylated interferon, nitazoxanide or an analog thereof, a compound of formula A, or a nucleoside analog,

formula A

Preferably, the nucleoside analogue is selected from entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide.

6. The use of claim 5, wherein the nucleoside analog is selected from the group consisting of entecavir, tenofovir disoproxil fumarate, and tenofovir alafenamide.

7. The use of any one of claims 1-6, wherein 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.

8. The use of claim 7, wherein the medicament is formulated for oral administration, preferably in the form of a tablet or capsule.

9. A pharmaceutical composition for treating or preventing viral hepatitis comprising a therapeutically effective amount of fenretinide or a pharmaceutically acceptable salt thereof and optionally one or more additional therapeutic or prophylactic agents selected from at least one of an interferon, a PEGylated interferon, nitazoxanide or an analog thereof, a compound of formula A or a nucleoside analog, and a pharmaceutically acceptable carrier,

formula A

Preferably, the nucleoside analogue is selected from entecavir, tenofovir disoproxil fumarate and tenofovir alafenamide.

10. The use of claim 1, wherein the derivative of compound 1 is deuterated fenretinide.

Images