WO2020259706A1 - 氨来占诺用于制备抗肝炎病毒药物的用途 - Google Patents

氨来占诺用于制备抗肝炎病毒药物的用途 Download PDF

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WO2020259706A1
WO2020259706A1 PCT/CN2020/098635 CN2020098635W WO2020259706A1 WO 2020259706 A1 WO2020259706 A1 WO 2020259706A1 CN 2020098635 W CN2020098635 W CN 2020098635W WO 2020259706 A1 WO2020259706 A1 WO 2020259706A1
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hepatitis
day
virus
hepatitis virus
drug
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PCT/CN2020/098635
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English (en)
French (fr)
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王鑫
杨金波
徐锡明
李俪
赵晨阳
王宇欣
武娟
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青岛海洋生物医药研究院股份有限公司
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Priority to EP20831767.7A priority Critical patent/EP3991729A4/en
Priority to KR1020227000310A priority patent/KR20220018552A/ko
Publication of WO2020259706A1 publication Critical patent/WO2020259706A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of medicine, in particular to the use of amlexinol for preparing anti-hepatitis virus drugs.
  • Hepatitis virus infection often causes viral hepatitis, and related pathogens mainly include hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV) and hepatitis G virus (HGV), among which the first four have a wide range of infections and are more harmful.
  • Therapeutic drugs for hepatitis virus infection are diverse, and the mode of action is mainly to inhibit the virus-encoded DNA polymerase during the reverse transcription process of virus replication, or to destroy the stability of virus-encoded RNA during the assembly process of virus particles, or by acting on the host. Interferon receptors indirectly inhibit virus replication.
  • DNA polymerase inhibitors are mainly nucleoside or nucleotide analogues, such as lamivudine, entecavir, and Ade Fovir and tenofovir, these nucleoside or nucleotide analogs inhibit viral DNA synthesis by inhibiting viral polymerase, thereby achieving antiviral effects.
  • Long-term use of lamivudine, adefovir, entecavir, etc. may cause hepatitis B virus mutation.
  • DNA polymerase inhibitors cannot directly inhibit the expression of hepatitis B surface antigen (HBsAg).
  • HBsAg-based vaccines cannot produce HBsAg antibodies.
  • Some drugs that are undergoing clinical trials are targeted at viral HBcAg and viral RNA, or neutralize viral surface antigen (HBsAg) through specific antibodies. The ultimate effect of these drugs is unknown.
  • RNA interference RNA interference
  • Interferon is also commonly used in the treatment of hepatitis B. It implements an indirect anti-hepatitis B infection effect through the immune system.
  • the target is interferon receptors, including short-acting interferons and long-acting interferons.
  • PEGASYS is a long-acting interferon used to treat hepatitis B and C.
  • Interferon has obvious side effects and can cause high fever, and usually requires injections. Patient compliance is poor. At the same time, it cannot be directly and effectively reduced in patients with high HBsAg. In addition, for patients with hepatitis B infection with liver fibrosis, interferon therapy is usually not recommended.
  • the antiviral drug is a drug for preventing and/or treating hepatitis virus infection or a drug for preventing and/or treating diseases caused by hepatitis virus infection.
  • the virus is a DNA virus and/or an RNA virus, preferably hepatitis B virus.
  • the disease caused by hepatitis virus infection is acute hepatitis, chronic hepatitis, cirrhosis, liver fibrosis, liver cancer, liver damage and/or other medically defined diseases.
  • the forms of amlexinol include pharmaceutically acceptable salts, hydrates, solvates or prodrugs, as long as the pharmaceutically acceptable salts, hydrates, solvates or precursors
  • the drug also has a similar anti-hepatitis virus effect.
  • a pharmaceutical composition which comprises amlexinol and another one or more antiviral active substances, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is composed of amlexinol, another one or more antiviral active substances and a pharmaceutically acceptable carrier.
  • the other one or more antiviral active substances are transcription or reverse transcription inhibitors, DNA polymerase inhibitors, reverse transcriptase inhibitors, capsid assembly regulators, capsid protein inhibitors, viral mRNA inhibitors, or combinations thereof.
  • the other one or more antiviral active substances are nucleoside or nucleotide analogs, preferably lamivudine, zidovudine, stanvudine, tenofovir, One or more of Entecavir, Abacavir, Zalcitabine, Emtricitabine, Didanosine, Nevirapine, Delavirdine, Efavirenz, Etravirin, and Ripavirin , More preferably, is one or more of lamivudine, tenofovir or entecavir.
  • the pharmaceutical composition for preparing drugs for preventing and/or treating viral infections or drugs for preventing and/or treating diseases caused by viral infections is provided.
  • the virus is a DNA virus and/or RNA virus, more preferably, hepatitis B virus.
  • the disease caused by viral infection is acute hepatitis, chronic hepatitis, cirrhosis, liver fibrosis, liver cancer, and/or other medically defined diseases.
  • the antiviral drug is an oral preparation or an injection preparation; preferably, the oral preparation is a tablet, capsule, granule and/or oral liquid.
  • the fourth aspect of the present invention there is provided a method for preventing and/or treating viral infections or preventing and/or treating diseases caused by viral infections, specifically, administering ammonia or the said Pharmaceutical composition (as described in the second aspect).
  • the virus is a DNA virus or an RNA virus, preferably, hepatitis B virus.
  • the subject is a human.
  • the diseases caused by hepatitis virus infection are acute hepatitis, chronic hepatitis, cirrhosis, liver cancer, liver damage, and/or other medically defined diseases in the near sense.
  • the method of administration is oral.
  • the dosage of the method is 1 to 500 mg/day, preferably, 50 to 500 mg/day; most preferably, 50 to 200 mg/day.
  • the method includes administration once or multiple times a day (such as 1, 2, 3, or 4 times a day), or once multiple days (such as once every 1, 2 or 3 days) .
  • a day such as 1, 2, 3, or 4 times a day
  • once multiple days such as once every 1, 2 or 3 days
  • the method is administered one or more times a day (such as 1, 2, 3, or 4 times a day), and the total daily dosage is 1 to 500 mg/day, preferably 50 to 500 mg/day. Day; best, 50 ⁇ 200mg/day.
  • a method for non-therapeutic inhibition of viral infection in vitro comprises the steps of: culturing cells that have been transfected by the virus (such as those that have been ⁇ 24 hours of cells), thereby inhibiting viral infection.
  • the virus is as defined above.
  • culture for 24 to 144 hours.
  • Figure 1 shows the inhibition of HBV genomic DNA replication in a cell model by Amlexinol.
  • Figure 2 shows that Amlexinol reduces the level of hepatitis B virus surface antigen in a cell model.
  • Figure 3 shows that Amlexinol reduces the level of hepatitis B virus surface antigen in a mouse model of venous hypertension.
  • Figure 4 shows that Amlexinol reduces the level of hepatitis B virus core antigen in a mouse model of venous hypertension.
  • Figure 5 shows that Amlexinol reduces hepatitis B virus DNA replication in a mouse model of intravenous hypertension.
  • Figure 6 shows that Amlexinol combined with clinically used nucleotide analog drugs reduces the level of hepatitis B virus surface antigen in a cell model.
  • Figure 7 shows the dose-effect relationship of Amlexinol in inhibiting hepatitis B virus in a mouse model of venous hypertension.
  • Figure 8 shows the inhibitory effect and dose-effect relationship of amlexinol in inhibiting subtype A hepatitis B virus in a mouse model of venous hypertension.
  • Figure 9 shows the effect of amlexinol on the innate immunity of mice.
  • Figures 10, 11, and 12 show that Amlexinol inhibits hepatitis B virus replication in a HepAD38 cell model.
  • Figure 13 shows that Amlexinol reduces hepatitis B virus surface antigen levels in a cell model.
  • Figure 14 shows that Amlexinol reduces the level of hepatitis B virus core antigen in a cell model (HepAD38 cells).
  • Figure 15 shows the dose-effect relationship of Amlexinol in inhibiting hepatitis B virus in a mouse model of venous hypertension.
  • Figure 16 shows that amlexinol reduces the degree of liver damage in a mouse model of venous hypertension.
  • Figure 17 shows the inhibitory effect of amlexinol on 3TC/ETV resistant HBV.
  • Figure 18 shows that Amlexinol reduces hepatitis B surface antigen in a transgenic mouse model.
  • Figure 19 shows that amlexinol does not inhibit the number of immune cells in mice.
  • the methods and techniques of the present invention are usually performed according to traditional methods known in the art, unless otherwise specified.
  • the nomenclature, experimental methods and techniques related to biology, pharmacology, and medicine and medicinal chemistry described herein are known and commonly used in the art.
  • the chemical synthesis method, chemical analysis method, pharmaceutical preparation method, blending method and transmission method, and detection or testing method all adopt standard technology.
  • Amlexanox (English name Amlexanox, abbreviated as AMLE), is clinically used to treat oral ulcers, bronchial asthma, etc. There are no reports of anti-liver viral infections. Amlexanox was originally developed by Takeda Pharmaceuticals, Japan, and its mechanism of action is unknown. It was used in the form of oral tablets for the treatment of bronchial asthma at the initial stage of development, and was included in the Japanese Pharmacopoeia. Later, it was developed to resist oral ulcers and was widely used in clinical practice in European and American countries. In my country, Amlexanox is approved as a paste for the treatment of recurrent oral ulcers.
  • Amlexinol was found to have the effect of inhibiting TBK1/IKK ⁇ , and clinical trials for its use in the treatment of gastric ulcer, obesity, and non-alcoholic fatty liver are ongoing.
  • the anti-hepatic virus effect of Amlexanox has not been reported.
  • amlexanox The structural formula of amlexanox is shown in formula I:
  • compound of the invention refers to a compound represented by Formula I.
  • the term also includes pharmaceutically acceptable salts of compounds of formula I.
  • amlexinol includes the compound of formula I and pharmaceutically acceptable salts thereof.
  • salts refers to the salt formed by the reaction of the compound of the above formula I with inorganic acid, organic acid, alkali metal or alkaline earth metal and the like.
  • these salts include but are not limited to: (1) salts formed with the following inorganic acids: such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts formed with the following organic acids, such as acetic acid, lactic acid , Citric acid, succinic acid, fumaric acid, gluconic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, succinic acid, tartaric acid, maleic acid, or arginine; (3) Other salts, including salts with alkali metals or alkaline earth metals (such as sodium, potassium
  • prodrug means that when taken by a proper method, the precursor of the compound undergoes metabolism or chemical reaction in the patient's body to transform into a compound included in the general formula of the invention, and a salt or solution composed of the compound.
  • the precursor of the compound includes, but is not limited to, the carboxylic acid ester, carbonate, phosphate, nitrate, sulfate, sulfone ester, sulfoxide ester, amino compound, carbamate, azo compound, phosphoramide of the compound , Glucoside, ether, acetal and other forms.
  • therapeutically effective dose refers to any amount of the drug as described below, when used alone or in combination with another therapeutic agent, the amount of the drug can promote the regression of the disease, which is manifested as a reduction in the severity of disease symptoms , Increase the frequency and duration of the asymptomatic period, or prevent obstacles or disability caused by illness.
  • the "therapeutically effective dose” of the drug of the present invention also includes the “preventively effective dose”.
  • the “preventively effective dose” is any amount of the drug as described below, when the amount of the drug is administered alone or in combination with another therapeutic agent When a subject is at risk of developing a disease or suffering from a recurrence of the disease, the occurrence or recurrence of the disease can be suppressed.
  • the term “about” means that the value can vary from the recited value by no more than 1%.
  • the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “substantially consisting of” or “consisting of”.
  • the effective in vivo dosage and specific mode of administration will depend on the type, weight and age of the mammal being treated, the specific compound used and the specific use of these compounds.
  • the purpose changes.
  • Those skilled in the art can determine the effective dose level (ie, the dose level necessary to achieve the desired effect) according to conventional pharmacological methods.
  • the human clinical application of the product starts from a lower dosage level, and then continues to increase the dosage level until the desired effect is achieved.
  • acceptable in vitro studies can be used by existing pharmacological methods to establish useful doses and routes of administration of the compositions identified by this method.
  • the pharmaceutical composition of the main active ingredient can be used to treat, prevent and alleviate hepatitis virus infection and/or diseases caused by hepatitis virus infection.
  • the compounds of the present invention can be used to treat the following diseases: acute hepatitis, chronic hepatitis, liver cirrhosis, liver cancer, liver damage, or other medically defined diseases in a similar sense.
  • the pharmaceutical composition of the present invention contains the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier within a safe and effective amount.
  • the "safe and effective amount” refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 1-2000 mg of the compound/agent of the present invention, preferably, 10-500 mg (more preferably, 25-200 mg, such as 25-100 or 50-200 mg) of the compound/agent.
  • the "one dose” is a capsule or tablet.
  • the compounds of the present invention may also include one or more other antiviral active substances (for example, those as defined above).
  • “Pharmaceutically acceptable carrier” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and sufficiently low toxicity. "Compatibility” here means that the components in the composition can be blended with the compound of the present invention and between them without significantly reducing the efficacy of the compound.
  • pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, and solid lubricants (such as stearic acid).
  • Magnesium stearate calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween) ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • vegetable oils such as soybean oil, sesame oil, peanut oil, olive oil, etc.
  • polyols such as propylene glycol, glycerin, mannitol, sorbitol, etc.
  • emulsifiers such as Tween
  • wetting agents such as sodium lauryl sulfate
  • coloring agents such as sodium lauryl sulfate
  • flavoring agents such as pepperminophen, sorbitol, etc.
  • antioxidants
  • the method of administration of the compound or pharmaceutical composition of the present invention is not particularly limited.
  • Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration .
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with the following ingredients: (a) fillers or compatibilizers, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectant, For example, glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators, such as quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin;
  • the dosage form may also contain buffering agents.
  • Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared with coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifying agents, and the active compound or the release of the compound in such a composition may be released in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microcapsules with one or more of the above-mentioned excipients. 2) Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-Butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • the composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.
  • the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • the composition for parenteral injection may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile for reconstitution into sterile injectable solutions or dispersions. powder.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the dosage form of the compound of the present invention for topical administration includes ointment, powder, patch, spray and inhalant.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.
  • the compound of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.
  • a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage is the pharmaceutically effective dosage considered to be administered.
  • the daily administration dose is usually 1 to 2000 mg, preferably 20 to 500 mg (more preferably 50 to 200 mg).
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are within the skill range of a skilled physician.
  • Ameliacinol can be used alone or in combination with other related antiviral drugs, the effect is better than existing anti-hepatitis virus drugs, and it has a good commercial prospect.
  • Example 1 Amlexinol inhibits hepatitis B virus genomic DNA replication in a cell model.
  • HBV viral genomic DNA (genotype B), virus-infected cells established model. 16-24 hours after transfection, add amlexanol, lamivudine, entecavir or tenofovir at a final concentration of 10 ⁇ M.
  • Example 2 Amlexinol reduces the level of hepatitis B virus surface antigen in a cell model, and its effect is better than existing clinical drugs.
  • hepatocyte HepG2 cells at 10 6 cells / well were seeded in 6-well plates, overnight, using lipofectamine 2000 transfected per well of 2 ⁇ g linear HBV viral genomic DNA (genotype B), virus-infected cells established model. 16-24 hours after transfection, add amlexanol, lamivudine, entecavir or tenofovir at a final concentration of 10 ⁇ M. The control group was added with equal volume of DMSO (1/1000, v/v). 48 hours after transfection, the cells in each well were passaged on a 6cm culture plate. 96 hours after transfection, the level of hepatitis B surface antigen (HBsAg) in the culture supernatant was detected and quantified by ELISA.
  • HBsAg hepatitis B surface antigen
  • Example 3 Amlexinol reduces the level of hepatitis B virus surface antigen in a mouse model of venous hypertension.
  • Wild-type mice (C57/B6) were injected with 10 ⁇ g of hepatitis B virus genomic DNA (genotype B) in a short period of time through tail vein injection.
  • the venous hypertension model was used to establish viral infection and randomly grouped into groups of 5 mice. From the second day onwards, oral administration of amlexanol (AMLE, 50mg/kg/day), or lamivudine (530mg/kg/day), or an equal volume and concentration of cosolvent cyclodextrin (control) .
  • HBsAg hepatitis B virus surface antigen
  • amlexanol reduces the level of hepatitis B virus surface antigen in a mouse model of intravenous hypertension, and its effect is better than lamivudine.
  • Example 4 Amlexinol reduces the level of hepatitis B virus core antigen in a mouse model of venous hypertension, and its effect is equivalent to that of lamivudine.
  • Wild-type mice (C57/B6) were injected with 10 ⁇ g of hepatitis B virus genomic DNA (genotype B) in a short period of time through tail vein injection.
  • the venous hypertension model was used to establish viral infection and randomly grouped into groups of 5 mice. From the second day, oral administration of amlexinol (AMLE, 50mg/kg/day), or lamivudine (530 mg/kg/day), or an equal volume and concentration of cosolvent cyclodextrin (control ).
  • hepatitis B virus core antigen HBeAg
  • amlexanol reduces the level of hepatitis B virus core antigen in a mouse model of intravenous hypertension, and its effect is equivalent to lamivudine.
  • Example 5 Amlexinol reduces hepatitis B virus DNA replication in a mouse model of venous hypertension.
  • Wild-type mice (C57/B6) were injected with 10 ⁇ g of hepatitis B virus genomic DNA (genotype B) in a short period of time through tail vein injection.
  • the venous hypertension model was used to establish viral infection and randomly grouped into groups of 5 mice. From the second day onwards, oral administration of amlexanol (AMLE, 50mg/kg/day), lamivudine (530mg/kg/day), entecavir (0.03mg/kg/day) or tenofovir (530mg/kg/day), or an equal volume of cyclodextrin (control).
  • ALE amlexanol
  • lamivudine 530mg/kg/day
  • entecavir entecavir
  • tenofovir 530mg/kg/day
  • control an equal volume of cyclodextrin
  • AMLE can significantly inhibit viral DNA load, and its effect is no less than that of clinically used nucleotide-like drugs such as lamivudine and entecavir.
  • Example 6 Amlexanol can be combined with clinically used nucleotide analog drugs to reduce the level of hepatitis B virus surface antigen in a cell model.
  • HBV viral genomic DNA (genotype B). 16-24 hours after transfection, add the compound with a final concentration of 10 ⁇ M as shown in the figure ("+" represents a certain drug or active substance). The control group was added with equal volume of DMSO (1/1000, v/v). 48 hours after transfection, the cells in each well were passaged on a 6cm culture plate. 96 hours after transfection, the HBV surface antigen (HBsAg) in the culture medium was detected by ELISA.
  • HBV surface antigen HBsAg
  • Example 7 The dose-effect relationship of Alexandrol inhibiting hepatitis B virus in a mouse model of venous hypertension.
  • Wild-type mice (C57/B6) were injected with 10 ⁇ g of hepatitis B virus genomic DNA (genotype B) in a short period of time through tail vein injection.
  • the venous hypertension model was used to establish viral infection and randomly grouped into groups of 5 mice. From the second day onwards, the oral administration of amrazanoic acid was given by oral gavage. The daily dosage is as shown in Figure 7, or the cosolvent cyclodextrin (control) with an equal volume and an equal concentration.
  • HBsAg HBV surface antigen
  • Example 8 The inhibitory effect and dose-effect relationship of Alenezinol on the hepatitis B virus of subtype A in a mouse model of venous hypertension.
  • Wild-type mice (C57/B6) were quickly injected with viral genome DNA (genotype A) 10 ⁇ g/mouse through tail vein injection in a short period of time.
  • Virus infection was established using a venous hypertension model and randomly grouped. From the second day after injection, lamivudine (3TC, 530mg/kg/day, once/day) and tenofovir (TD, 530mg/kg/day) were administered orally according to the compound drug-time curve. 1 time/day), or the designated concentration of Ameliacinol (4, 8, 16mg/kg/time, 2 times/day) or an equal volume of cyclodextrin (NC). On the 9th day after infection, about 100 ⁇ l of blood was collected from the tail, the serum was separated, and the concentration of HBV core antigen (HBeAg) in the peripheral blood was detected by ELISA.
  • HBV core antigen HBV core antigen
  • Example 9 The effect of amlexinol on the innate immunity of mice.
  • Wild-type mice (C57/B6) were quickly injected with viral genome DNA (genotype A) 10 ⁇ g/mouse through tail vein injection in a short period of time. Virus infection was established using a venous hypertension model and randomly grouped. From the next day, according to the compound drug-time curve, oral administration of lamivudine (3TC, 530mg/kg/day, once/day) and tenofovir (TD, 530mg/kg/day, once /Day), or the designated concentration of Ammonazinol (4, 8, 16mg/kg/time, 2 times/day) or the same volume and concentration of the cosolvent cyclodextrin (NC). On the 9th day after infection, about 100 ⁇ l of blood was collected from the tail, the serum was separated, and the concentration of type I interferon IFN- ⁇ in the peripheral blood of the mouse was detected by ELISA.
  • lamivudine 3TC, 530mg/kg/day, once/day
  • TD
  • Example 10 The effect of Alexandrol on hepatitis B virus in HepAD38 cell model
  • Lysis buffer A 50mM Tris-HCl, pH 7.4, 1mM EDTA, and 1% NP-40
  • the supernatant and precipitate of the lysate were collected, and DNA was extracted from it. And quantified by quantitative PCR.
  • the DNA in the precipitation of the cell lysate was extracted and the linear DNA was removed by exonuclease (NEB), so only the circular DNA (cccDNA) was checked ( Figure 10).
  • the viral DNA in the supernatant of the cell lysate is defined as core particle DNA ( Figure 11). Refer to Figure 12 for the quantification of viral DNA in the cell culture supernatant.
  • Example 11 The effect of ammonia on the surface antigen and core antigen of hepatitis B virus in a cell model.
  • Example 12 The effect of amlexinol in a mouse model of venous hypertension
  • 6-week-old male C57 mice were injected with 10 ⁇ g of viral genome DNA (genotype A) in a short period of time through tail vein injection, and the venous hypertension model was used to establish viral infection and randomly grouped. From the second day after injection, according to the compound drug-time curve, oral administration of Entecavir (ETV, 0.03mg/kg/day, 1 time/day), or a specified concentration of ammonia (equivalent to the adult dose 25 , 50, 100, 150, 200 mg/day, 1 time/day) or an equal volume of solvents (model). At the same time, a blank control was established, and the oral administration of uninfected mice was equivalent to the model group. The grouping and the dosage of each group of animals are shown in the following table:
  • 6-week-old male C57 mice were injected with 10 ⁇ g of viral genome DNA (genotype A) in a short period of time through tail vein injection, and the venous hypertension model was used to establish viral infection and randomly grouped.
  • oral administration of Entecavir ETV, 0.03mg/kg/day, 1 time/day
  • lamivudine 3TC, 530mg/kg/day, 1 time) /Day
  • Tenofovir TDF, 530mg/kg/day, 1 time/day
  • Amaloxanol equivalent to an adult dose of 150mg/day, 1 time/day
  • an equal volume of solvent model.
  • a blank control was established, and the oral administration of uninfected mice was equivalent to the model group.
  • the grouping and the dosage of each group of animals are shown in the following table:
  • mice in each group were given an appropriate amount of blood from the tail vein, and on the 9th day after the mice were sacrificed, the remaining peripheral blood was taken. All blood samples were centrifuged to collect serum, and the concentration of HBV surface antigen (HBsAg) was detected by ELISA.
  • the specific method refers to the ELISA kit instructions.
  • Example 14 The effect of Alexandrol in a transgenic mouse model
  • mice 10 male C57BL/6-HBV transgenic mice (provided by Beijing Weitongda Biotechnology Co., Ltd.) at 6 weeks of age were randomly divided into 2 groups (5 in the control group and 5 in the administration group).
  • the dosage of each group of animals is as follows:
  • Route of administration intragastrically, 1mL sterile syringe is used for administration.
  • Dosing frequency The test product is administered once a day for 35 consecutive days, with free diet and water.
  • Dosing capacity 10mL/kg, according to the last weight of each animal before dosing, determine the dosage of each animal.
  • HBV surface antigen HBV surface antigen
  • the inhibition rate is calculated according to the following formula:
  • Inhibition rate [(OD450 control hole-OD450 administration hole)/OD450 control hole] ⁇ 100%
  • HBV transgenic mouse model was used to evaluate the efficacy of Amalizanol.
  • the detection results of HBV surface antigen HBsAg are shown in Figure 18.
  • Amalizanol can inhibit HBsAg in transgenic mice. Level.
  • 6-week-old male C57 mice were injected with 10 ⁇ g of viral genome DNA (genotype A) in a short period of time through tail vein injection, and the venous hypertension model was used to establish viral infection and randomly grouped.
  • lamivudine (3TC, 530mg/kg/day, once/day) and tenofovir (TDF, 530mg/kg/day, 1 time/day), Entecavir (ETV, 0.03mg/kg/day, 1 time/day), or specified concentration of amlexanolide (4, 8, 16mg/kg/time, 2 times/day) or equal volume, etc.
  • Concentration solvent model
  • a blank control was established, and the oral administration of uninfected mice was equivalent to the model group.
  • the grouping and the dosage of each group of animals are as follows:
  • mice On the 9th day, the mice were sacrificed and blood was collected, and then stained with flow cytometry antibody after redness. All cells were treated with anti-Fc ⁇ RIII/II (purchased from BD Biosciences) in PBS for 30 minutes.
  • CD3e-FITC purchased from BD Biosciences.
  • CD4-PE, CD8a-APC-Vio770 and CD34-FITC (GE).
  • CD19-APC, CD11b-PE-Cyanine7 and CD117-efflor 450 purchased from eBioscience. After staining, the sample was suspended in 500 ⁇ L PBS and analyzed by BD AriaIII flow cytometer.
  • Amlexanox has an inhibitory effect on both B cells and T cells, and the inhibitory effect is dose-dependent.
  • lamivudine also has a certain inhibitory effect on immune cells. As the concentration increases, the suppression of immune cells increases, but the inhibitory effect on HBV increases, which also shows that ammonia does not exert its anti-HBV effect through the immune system.
  • the MTT method was used to test the toxicity of ammonia in HepG2, HCT-116, Hela, A549, MCF7, MKN45, THP-1, Huh7 and 293 cells. Inoculate various cells in the logarithmic growth phase in 96-well plates at 6,000 cells/well. After culturing for 24 hours, add different concentrations of Ammonacinol (final concentrations are 100, 50, 25, 12.5, 6.25, 3.125 ⁇ M) , The positive control doxorubicin hydrochloride (final concentration is 1 ⁇ M), the blank control group plus an equal volume of culture medium, the solvent control group DMSO dosage is subject to 0.1% of the maximum dose used in the test group, each concentration set 4 hole.
  • the inhibition rate of tumor cell growth is calculated according to the following formula:
  • Inhibition rate (%) [(OD570 control well-OD570 administration well)/OD570 control well] ⁇ 100.
  • the compound of the present invention has no obvious cytotoxicity in the 9 cells tested at a concentration below 50 ⁇ M. At a concentration of 100 ⁇ M, there is a certain degree of cytotoxicity in MKN45, HCT-116, MCF7 and Huh7 cells, and no obvious cytotoxicity is seen in the other 5 cells.

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Abstract

本发明提供了氨来占诺用于制备抗肝炎病毒药物的用途。具体地，本发明提供了氨来占诺用于制备抗病毒药物的新用途，特别是用于制备抗肝炎病毒的用途。氨来占诺作为抗肝炎病毒药物，既可以单独使用也可以与其它抗肝炎病毒药物联用。

Description

氨来占诺用于制备抗肝炎病毒药物的用途 技术领域
本发明涉及医药领域，具体地涉及氨来占诺用于制备抗肝炎病毒药物的用途。
背景技术
肝炎病毒感染往往引起病毒性肝炎，相关的病原体主要包括甲型肝炎病毒(HAV)、乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、丁型肝炎病毒(HDV)、戊型肝炎病毒(HEV)和庚型肝炎病毒(HGV)等，其中前四种感染范围较广，危害较大。针对肝炎病毒感染的治疗药物多样，作用的方式主要是抑制病毒复制的逆转录过程中病毒编码的DNA聚合酶，或者破坏病毒粒子组装过程中病毒编码的RNA的稳定性、或者通过作用于宿主的干扰素受体间接抑制病毒复制。
以乙型肝炎病毒为例，临床常用的药物以DNA聚合酶抑制剂居多，目前上市的DNA聚合酶抑制剂主要是核苷或核苷酸类似物，例如：拉米夫定、恩替卡韦、阿德福韦和替诺福韦等，这些核苷或核苷酸类似物通过抑制病毒聚合酶来抑制病毒DNA合成，从而达到抗病毒的效果。拉米夫定、阿德福韦、恩替卡韦等长期服用，具有导致乙肝病毒变异的可能。而且DNA聚合酶抑制剂并不能直接抑制乙肝表面抗原(HBsAg)的表达。
高HBsAg水平在慢性乙肝病毒感染病人中会诱导T细胞耗竭，采用基于HBsAg为基础的疫苗并不能产生HBsAg抗体。针对这些药物的缺陷和乙肝感染的特点在临床治疗当中，往往在血清HBsAg水平阴性的情况下给予病人注射疫苗，并提高获得HBsAg抗体的可能。因此，降低HBsAg水平成为治疗慢性乙肝病毒感染的有效途径。部分正在进行临床试验的药物，其针对的靶点包括病毒的HBcAg和病毒的RNA，或者通过特异性抗体中和病毒的表面抗原(HBsAg)，这些药物的最终效果未知。比如，目前Arrowhead制药公司在使用以在研药物JNJ-3989为代表的RNA干扰(RNAi)技术，最高可降低99％的HBsAg，但是RNAi技术需要较好的药物传递系统，和严格的储存技术，且无法口服，成本较高。
干扰素在乙肝治疗中也较为常用，它是通过免疫系统实施间接的抗乙肝感染作用，靶点是干扰素受体，包括短效干扰素和长效干扰素。如派罗欣就是一种用于治疗乙型和丙型肝炎的长效干扰素。干扰素副作用明显，会引起高烧等，且通常需要注射给药，病人依从性较差，同时也无法在高HBsAg病人中直接有效降低 其水平。另外，对于伴随肝纤维化的乙肝感染者，通常不推荐使用干扰素治疗。
发明内容
在本发明的第一方面，提供了氨来占诺用于制备抗病毒药物的新用途。
在一些方案中，所述抗病毒药物为预防和/或治疗肝炎病毒感染的药物或者是预防和/或治疗由于肝炎病毒感染所导致的疾病的药物。
在一些优选方案中，所述病毒为DNA病毒和/或RNA病毒，较优地，为乙肝病毒。
在另一些优选方案中，所述由于肝炎病毒感染所导致的疾病为急性肝炎、慢性肝炎、肝硬化、肝纤维化、肝癌、肝脏损伤和/或其它医学上的近义界定疾病。
在一些方案中，氨来占诺存在的形式，包括药学上可接受的盐、水合物、溶剂化物或前体药物，只要所述的药学上可接受的盐、水合物、溶剂化物或前体药物也具有类似的抗肝炎病毒作用。
在本发明的第二方面，提供了一种药物组合物，其包含氨来占诺和另外一种或多种抗病毒活性物质，以及药学上可接受的载体。
在一些方案中，所述药物组合物，由氨来占诺和另外一种或多种抗病毒活性物质以及药学上可接受的载体构成。
在一些优选方案中，所述另外一种或多种抗病毒活性物质为转录或逆转录抑制剂、DNA聚合酶抑制剂、逆转录酶抑制剂、衣壳组装调节剂、壳蛋白抑制剂、病毒mRNA抑制剂，或它们的组合。
优选地，所述另外一种或多种抗病毒活性物质为核苷或核苷酸类似物，较优地，为拉米夫定、齐多夫定、司坦夫定、替诺福韦、恩替卡韦、阿巴卡韦、扎西他滨、恩曲他滨、地丹诺辛、奈韦拉平、地拉韦啶、依非韦伦、依曲韦林、利匹韦林中的一种或多种，更优地，为拉米夫定、替诺福韦或恩替卡韦的中的一种或多种。
在本发明的第三方面，提供了所述药物组合物用于制备预防和/或治疗病毒感染的药物或者是预防和/或治疗由于病毒感染所导致疾病的药物的用途。
优选地，所述病毒为DNA病毒和/或RNA病毒，较优地，为乙肝病毒。
在一些方案中，所述由于病毒感染所导致的疾病为急性肝炎、慢性肝炎、肝硬化、肝纤维化、肝癌和/或其它医学上的近义界定疾病。
在另一些优选方案中，所述抗病毒药物为口服制剂或注射制剂；较优地，所 述的口服制剂为片剂、胶囊剂、颗粒剂和/或口服液。
在本发明的第四方面，提供了一种预防和/或治疗病毒感染或者是预防和/或治疗由于病毒感染所导致的疾病的方法，具体为对受试者施用氨来占诺或者所述药物组合物(如第二方面所述)。
优选地，所述病毒为DNA病毒或RNA病毒，较优地，为乙肝病毒。
优选地，所述受试者为人。
所述由于肝炎病毒感染所导致的疾病为急性肝炎、慢性肝炎、肝硬化、肝癌、肝脏损伤，和/或其它医学上的近义界定疾病。
优选地，所述方法的施用方式为口服。
优选地，所述方法的施用剂量为1～500mg/日，较佳地，50～500mg/日；最佳地，50～200mg/日。
优选地，所述的方法包括每日一次或多次地施用(如每日1、2、3或4次施用)、或者，多日一次地施用(如每1、2或3日施用一次)。
优选地，所述的方法每日一次或多次地施用(如每日1、2、3或4次施用)且每日总施用剂量为1～500mg/日，较佳地，50～500mg/日；最佳地，50～200mg/日。
在本发明的第五方面，提供了一种体外非治疗性抑制病毒感染的方法，包括步骤：在氨来占诺的存在下，培养已被病毒转染的细胞(如已被病毒转染16～24小时的细胞)，从而抑制病毒感染。
优选地，所述病毒如前定义。
优选地，培养24～144h。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明：
图1显示了氨来占诺在细胞模型中抑制乙肝病毒基因组DNA复制。
图2显示了氨来占诺在细胞模型中降低乙肝病毒表面抗原水平。
图3显示了氨来占诺在静脉高压小鼠模型中降低乙肝病毒表面抗原水平。
图4显示了氨来占诺在静脉高压小鼠模型中降低乙肝病毒核心抗原水平。
图5显示了氨来占诺在静脉高压小鼠模型中降低乙肝病毒DNA复制。
图6显示了氨来占诺与临床使用的核苷酸类似物药物联用，在细胞模型中降低乙肝病毒表面抗原水平。
图7显示了氨来占诺在静脉高压小鼠模型中抑制乙肝病毒的量效关系。
图8显示了氨来占诺在静脉高压小鼠模型中抑制A亚型乙肝病毒的效果及量效关系。
图9显示了氨来占诺对小鼠天然免疫的影响。
图10、11和12显示了氨来占诺在HepAD38细胞模型中抑制乙肝病毒复制。
图13显示了氨来占诺在细胞模型中降低乙肝病毒表面抗原水平。
图14显示了氨来占诺在细胞模型中(HepAD38细胞)降低乙肝病毒核心抗原水平。
图15显示了氨来占诺在静脉高压小鼠模型中抑制乙肝病毒的量效关系。
图16显示了氨来占诺在静脉高压小鼠模型中降低了肝损伤程度。
图17显示了氨来占诺对3TC/ETV耐药性HBV的抑制作用。
图18显示了氨来占诺在转基因鼠模型中降低了乙肝表面抗原。
图19显示了氨来占诺不抑制小鼠免疫细胞的数量。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现老药氨来占诺用于制备抗病毒药物的新用途。在此基础上，发明人完成了本发明。
术语说明
本发明的方法与技术通常依据本领域已知的传统方法进行，除非另有说明。与本文中描述的生物学、药理学、及医学与医药化学相关的命名法，及实验方法与技术是本领域已知且常用的。化学合成法、化学分析法、医药制法、调配法与传送法，及检测或测试法均采用标准技术。
除非另有说明，否则本文中所使用的科学与技术术语应具有那些本领域普通技术人员通常理解的含义。
氨来占诺及其抗病毒作用
氨来占诺(英文名Amlexanox，简写作AMLE)，临床用于治疗口腔溃疡、支气管哮喘等，该药未有抗肝脏病毒感染的报道。氨来占诺最初由日本武田制药开 发，作用机制未明。其在开发初期是以口服片剂的形式用于治疗支气管哮喘，并被收入日本药典，后被开发出抗口腔溃疡的功能，并在欧美国家被广泛的应用于临床。在我国，氨来占诺被批准以糊剂的形式用于治疗复发性口腔溃疡。在2013年，氨来占诺被发现具有抑制TBK1/IKKε的作用，其用于治疗胃溃疡、肥胖、非酒精性脂肪肝等临床试验正在进行中。氨来占诺的抗肝脏病毒作用未见报道。
氨来占诺的结构式如式I所示：
Figure PCTCN2020098635-appb-000001
如本文所用，术语“本发明化合物”指式I所示的化合物。该术语还包括及式I化合物的药学上可接受的盐。
在本发明中，术语“氨来占诺”包括式I化合物及其药学上可接受的盐。
如本文所用，术语“药学上可接受的盐”是指上述式I化合物与无机酸、有机酸、碱金属或碱土金属等反应生成的盐。这些盐包括但不限于：(1)与如下无机酸形成的盐：如盐酸、氢溴酸、氢碘酸、硫酸、硝酸、磷酸；(2)与如下有机酸形成的盐，如乙酸、乳酸、柠檬酸、琥珀酸、延胡索酸、葡萄糖酸、安息香酸、甲烷磺酸、乙烷磺酸、苯磺酸、对甲苯磺酸、草酸、丁二酸、酒石酸、马来酸、或精氨酸；(3)其它的盐，包括与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐，铵盐或水溶性的胺盐(如N-甲基葡糖胺盐)、低级的烷醇铵盐以及其它药学上可接受的胺盐(比如甲胺盐、乙胺盐、丙胺盐、二甲基胺盐、三甲基胺盐、二乙基胺盐、三乙基胺盐、叔丁基胺盐、乙二胺盐、羟乙胺盐、二羟乙胺盐、三羟乙胺盐，以及分别由吗啉、哌嗪、赖氨酸形成的胺盐)。
术语“前体药物”指当用适当的方法服用后，该化合物的前体在病人体内进行代谢或化学反应而转变成本发明通式所包含的化合物，以及化合物所组成的盐或溶液。化合物的前体包括但不局限于所述化合物的羧酸酯、碳酸酯、磷酸酯、硝酸酯、硫酸酯、砜酯、亚砜酯、氨基化合物、氨基甲酸盐、偶氮化合物、磷酰胺、葡萄糖苷、醚、乙缩醛等形式。
术语“治疗有效剂量”是指药物的任何如下所述的量，当单独使用或与另一种治疗剂组合使用该量的药物时，可促进疾病消退，疾病消退表现为疾病症状的严重度降低、无疾病症状期的频率和持续时间增加、或者防止由患病导致的障碍或 失能。本发明药物的“治疗有效剂量”也包括“预防有效剂量”，“预防有效剂量”是药物的任何如下所述的量，当将该量的药物单独施用或者与另一种治疗剂组合施用于具有发生疾病的风险或者遭受疾病复发的受试者时，可抑制疾病的发生或复发。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
在本发明中，如对本领域所属技术人员显而易见地，有效的体内给药剂量及具体的给药方式会根据所治疗的哺乳动物种类、体重和年龄，所使用的具体化合物及使用这些化合物的具体目的而变化。本领域所属技术人员根据常规的药理学方法可确定有效剂量水平(即达到所需效果所必需的剂量水平)。通常，产物的人体临床应用从较低的剂量水平开始，随后不断提高剂量水平直到达到所需的效果。可选择地，可通过现有的药理学方法采用可接受的体外研究来建立本方法鉴定的组合物的有用剂量和给药途径。
药物组合物和施用方法
由于本发明化合物如氨来占诺具有优异的抗病毒能力，因此本发明化合物及其各种晶型，药学上可接受的无机或有机盐，水合物或溶剂合物，以及含有本发明化合物为主要活性成分的药物组合物可用于治疗、预防以及缓解肝炎病毒感染和/或由于肝炎病毒感染所导致的疾病。根据现有技术，本发明化合物可用于治疗以下疾病：急性肝炎、慢性肝炎、肝硬化、肝癌、肝脏损伤，或其它医学上的近义界定疾病。
本发明的药物组合物包含安全有效量范围内的本发明化合物或其药理上可接受的盐及药理上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg本发明化合物/剂，较佳地，含有10-500mg(更佳地，含有25～200mg如25～100或50～200mg)本发明化合物/剂。较佳地，所述的“一剂”为一个胶囊或药片。本发明的化合物还可包括一种或多种其他种抗病毒活性物质(例如如前定义的那些)。
“药学上可以接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温
Figure PCTCN2020098635-appb-000002
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、瘤内、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。1)用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。2)用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水 山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。3)用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明化合物可以单独给药，或者与其他药学上可接受的化合物联合给药。
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选20～500mg(更优选50～200mg)。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明具有如下主要有益效果：
(a)氨来占诺具有显著的抗病毒作用；
(b)氨来占诺具有优良的安全性。
(c)氨来占诺既可以单独使用也可以与其它相关抗病毒药物联用，效果优于现有抗肝炎病毒药物，具有良好的商业前景。
下面结合具体实施，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1氨来占诺在细胞模型中抑制乙肝病毒基因组DNA复制。
生长状态良好的肝细胞HepG2细胞以10 6细胞/孔接种于6孔板，过夜后，使用lipofectamine 2000每孔转染2μg线性HBV病毒基因组DNA(基因型B)，建立病毒感染细胞模型。转染后16-24小时，分别加入终浓度为10μM的氨来占诺、拉米夫定、恩替卡韦或替诺福韦。对照组加入等体积DMSO(1/1000，v/v)。96小时后，细胞被分为两组，一组抽提基因组DNA，另一组利用HIRT萃取法抽提游 离DNA，并利用DNA外切酶(NEB)移除线性DNA。环状HBV DNA和基因组GAPDH被定量PCR检测。环状HBV DNA最高值被定义为100％(n＝5)。
从图1中的对比可以看出，氨来占诺在细胞模型中抑制乙肝病毒基因组DNA复制，效果与现有临床药物相当。
实施例2氨来占诺在细胞模型中降低乙肝病毒表面抗原水平，其效果优于现有临床药物。
生长状态良好的肝细胞HepG2细胞以10 6细胞/孔接种于6孔板，过夜后，使用lipofectamine 2000每孔转染2μg线性HBV病毒基因组DNA(基因型B)，建立病毒感染细胞模型。转染后16-24小时，分别加入终浓度为10μM的氨来占诺、拉米夫定、恩替卡韦或替诺福韦。对照组加入等体积DMSO(1/1000，v/v)。转染后48小时，每孔细胞传代于一个6cm培养板。转染后96小时，培养基上清中的乙肝表面抗原(HBsAg)水平被ELISA法检测定量。
从图2中对比可以看出，氨来占诺在细胞模型中降低乙肝病毒表面抗原水平，其效果优于现有临床药物。
实施例3氨来占诺在静脉高压小鼠模型中降低乙肝病毒表面抗原水平。
野生型小鼠(C57/B6)在短时间内通过尾静脉注射迅速推入乙肝病毒基因组DNA(基因型B)10μg/只，利用静脉高压模型建立病毒感染，并随机分组，每组5只。第二天起，口服灌胃给药氨来占诺(AMLE，50mg/kg/天)、或拉米夫定(530mg/kg/天)、或者等体积等浓度助溶剂环糊精(对照)。感染后第9天，割尾取血约100μl/只，分离血清，并利用ELISA法检测外周血中乙肝病毒表面抗原(HBsAg)的浓度，于酶标仪上测定450nm处的OD值。
从图3中对比结果可以看出，氨来占诺在静脉高压小鼠模型中降低乙肝病毒表面抗原水平，其效果优于拉米夫定。
实施例4氨来占诺在静脉高压小鼠模型中降低乙肝病毒核心抗原水平，其效果与拉米夫定相当。
野生型小鼠(C57/B6)在短时间内通过尾静脉注射迅速推入乙肝病毒基因组DNA(基因型B)10μg/只，利用静脉高压模型建立病毒感染，并随机分组，每组5只。第二天起，口服灌胃给药氨来占诺(AMLE，50mg/kg/天)、或拉米夫定(530 mg/kg/天)、或者等体积等浓度助溶剂环糊精(对照)。感染后第9天，割尾取血约100μl/只，分离血清，并利用ELISA法检测外周血中乙肝病毒核心抗原(HBeAg)的浓度，于酶标仪上测定450nm处的OD值。
从图4中对比结果可以看出，氨来占诺在静脉高压小鼠模型中降低乙肝病毒核心抗原水平，其效果与拉米夫定相当。
实施例5氨来占诺在静脉高压小鼠模型中降低乙肝病毒DNA复制。
野生型小鼠(C57/B6)在短时间内通过尾静脉注射迅速推入乙肝病毒基因组DNA(基因型B)10μg/只，利用静脉高压模型建立病毒感染，并随机分组，每组5只。第二天起，口服灌胃给药氨来占诺(AMLE，50mg/kg/天)、拉米夫定(530mg/kg/天)、恩替卡韦(0.03mg/kg/天)或替诺福韦(530mg/kg/天)、或等体积等浓度助溶剂环糊精(对照)。感染后第11天，处死小鼠，分离小鼠肝脏，DpnI移除外源DNA，荧光定量PCR法检测小鼠肝脏中HBV基因组DNA(n＝5)。
从图5中可见，在动物模型中，AMLE可显著抑制病毒DNA的载量，其效果不低于临床使用的核苷酸类似药物拉米夫定和恩替卡韦等。
实施例6氨来占诺可与临床使用的核苷酸类似物药物联用，在细胞模型中降低乙肝病毒表面抗原水平。
生长状态良好的肝细胞HepG2细胞10 6细胞/孔接种于6孔板，过夜后，使用Lipofectamine 2000每孔转染2μg线性HBV病毒基因组DNA(基因型B)。转染后16-24小时，分别加入如图所示终浓度为10μM的化合物(“+”代表含有某种药物或活性物质)。对照组加入等体积DMSO(1/1000，v/v)。转染后48小时，每孔细胞传代于一个6cm培养板。转染后96小时，培养基中的HBV表面抗原(HBsAg)被ELISA法检测。
从图6中可见，在细胞模型中，氨来占诺与拉米夫定或替诺福韦联用，效果优于AMLE单用，显著优于拉米夫定或替诺福韦单用。
实施例7氨来占诺在静脉高压小鼠模型中抑制乙肝病毒的量效关系。
野生型小鼠(C57/B6)在短时间内通过尾静脉注射迅速推入乙肝病毒基因组DNA(基因型B)10μg/只，利用静脉高压模型建立病毒感染，并随机分组，每组5只。第二天起，口服灌胃给药氨来占诺，每天给药剂量如图7中所示，或等体积 等浓度助溶剂环糊精(对照)。感染后第9天，割尾取血约100μl/只，分离血清，并利用ELISA法检测外周血中HBV表面抗原(HBsAg)的浓度。
从图7中对比可以看出，随着氨来占诺浓度增加，其在静脉高压小鼠模型中抑制乙肝病毒的能力增强，这种量效关系在10mg/kg的剂量以下最为明显、在10-50mg/kg的剂量时有显著效果。
实施例8氨来占诺在静脉高压小鼠模型中抑制A亚型乙肝病毒的效果及量效关系。
野生型小鼠(C57/B6)在短时间内通过尾静脉注射迅速推入病毒基因组DNA(基因型A)10μg/只，利用静脉高压模型建立病毒感染，并随机分组。注射后第二天起，根据化合物药时曲线，口服灌胃给药拉米夫定(3TC，530mg/kg/天，1次/天)、替诺福韦(TD，530mg/kg/天，1次/天)，或指定浓度氨来占诺(4、8、16mg/kg/次，2次/天)或等体积等浓度助溶剂环糊精(NC)。感染后第9天，割尾取血约100μl/只，分离血清，并利用ELISA法检测外周血中HBV核心抗原(HBeAg)的浓度。
从图8中对比可以看出，随着氨来占诺浓度增加，其在静脉高压小鼠模型中抑制乙肝病毒的能力增强，这种量效关系在4～8mg/kg的剂量间最为明显，16mg/kg仍然有效。
实施例9氨来占诺对小鼠天然免疫的影响。
野生型小鼠(C57/B6)在短时间内通过尾静脉注射迅速推入病毒基因组DNA(基因型A)10μg/只，利用静脉高压模型建立病毒感染，并随机分组。第二天起，根据化合物药时曲线，口服灌胃给药拉米夫定(3TC，530mg/kg/天，1次/天)、替诺福韦(TD，530mg/kg/天，1次/天),或指定浓度氨来占诺(4、8、16mg/kg/次，2次/天)或等体积等浓度助溶剂环糊精(NC)。感染后第9天，割尾取血约100μl/只，分离血清，并利用ELISA法检测小鼠外周血中I型干扰素IFN-α的浓度。
从图9可以看出，氨来占诺对HBV病毒感染引起的天然免疫应答，尤其是I型干扰素的诱导，没有明显影响。
实施例10氨来占诺在HepAD38细胞模型中对乙肝病毒的影响
将2x10 5生长状态良好的稳定表达HBV的HepAD38细胞种于胶原质 (collagen)预处理过的6孔板中，加入相应浓度的测试物：恩替卡韦(ETV)终浓度1μM和氨来占诺(AML)终浓度分别为20μM、10μM和5μM。同时设置两组对照，一组仅加溶剂(DMF)，一组仅加抗生素(四环素(Tetracycline))。所有组别都设置3个复孔。加入测试物96小时和144小时后分别取500μL细胞培养液上清用于提取上清的DNA，qPCR检测上清中的HBV DNA量。144h后，收集细胞，经裂解缓冲液(Lysis buffer)A(50mM Tris-HCl,pH 7.4,1mM EDTA,and 1％NP-40)裂解后，分别收集裂解液的上清和沉淀，从中提取DNA，并由定量PCR定量。其中细胞裂解液沉淀中的DNA抽提出后由DNA外切酶(NEB)移除线性DNA，因此仅检查了其中的环状DNA(cccDNA)(图10)。细胞裂解液上清中的病毒DNA被定义为核心颗粒DNA(core particle DNA)(图11)。细胞培养基上清中的病毒DNA的定量参见图12。
结果如图10、11和12所示，可见氨来占诺在HepAD38细胞模型中显著抑制了乙肝病毒复制。
实施例11氨来占诺在细胞模型中对乙肝病毒表面抗原和核心抗原的影响。
将2x10 5个生长状态良好的稳定表达HBV的HepAD38细胞种于胶原质预处理过的6孔板中，加入相应浓度的测试物：恩替卡韦(ETV)终浓度1μM和氨来占诺(AML)终浓度分别为20μM、10μM和5μM。同时设置两组对照，一组仅加溶剂(DMF)，一组仅加抗生素(四环素)。所有实验都设置3个复孔。加入测试物后24小时、48小时、96小时和144小时分别取500μL培养物上清用于ELISA检测HBV表面抗原(HBsAg)浓度(图13)。
将2x10 5个生长状态良好的稳定表达HBV的HepAD38细胞种于胶原质预处理过的6孔板中，加入相应浓度的测试物：恩替卡韦(ETV)终浓度1μM和氨来占诺(AML)终浓度分别为16μM、10μM、8μM、5μM、4μM、2.5μM和2μM。同时设置仅加溶剂的对照组(DMF)。所有实验都设置3个复孔。加入测试物后24小时、48小时、72小时、96小时和144小时分别取500μL培养物上清用于ELISA检测HBV核心抗原(HBeAg)浓度。(图14)
结果如图13和14所示，可见氨来占诺能够在HepAD38细胞模型中显著降低乙肝病毒表面抗原水平和核心抗原水平。
实施例12氨来占诺在静脉高压小鼠模型中的作用
6周龄雄性C57小鼠在短时间内通过尾静脉注射迅速推入病毒基因组DNA(基因型A)10μg/只，利用静脉高压模型建立病毒感染，并随机分组。注射后第二天起，根据化合物药时曲线，口服灌胃给药恩替卡韦(ETV，0.03mg/kg/天，1次/天)，或指定浓度氨来占诺(等量于成人用药剂量25、50、100、150、200mg/天，1次/天)或等体积等浓度溶剂(模型)。同时设立空白对照，未感染小鼠口服给药等同于模型组。分组及各组动物给药剂量如下表所示：
Figure PCTCN2020098635-appb-000003
*表示此处的剂量为换算成成人用药剂量后的值。换算方法如下：假设成人体重60kg，成人用药剂量/天＝小鼠用药剂量/kg/天*60kg/9.1，例如成人25mg/天等效于小鼠3.8mg/kg/天。
给药第1、3、7天时，各组小鼠尾静脉取适量的血，第9天小鼠处死后，取剩余外周血。所有血液样品离心取血清，分为两份。一份由ELISA检测HBV表面抗原(HBsAg)的浓度，一份由谷丙转氨酶试剂盒(比色法)检查谷丙转氨酶(ALT)的酶活(南京建成生物工程研究所)。
结果如图15和16所示。由图15可见连续给氨来占诺7天后，高于50mg/天的剂量能够显著降低小鼠体内表面抗原(HBsAg)水平，抑制HBV活性。由图16可见氨来占诺能够有效降低谷丙转氨酶活，说明了氨来占诺能够有效降低肝损伤水平，对静脉高压和HBV引起的肝脏损伤具有修复作用。
实施例13
6周龄雄性C57小鼠在短时间内通过尾静脉注射迅速推入病毒基因组DNA(基因型A)10μg/只，利用静脉高压模型建立病毒感染，并随机分组。注射 后第二天起，根据化合物药时曲线，口服灌胃给药恩替卡韦(ETV，0.03mg/kg/天，1次/天)、拉米夫定(3TC，530mg/kg/天，1次/天)、替诺福韦(TDF，530mg/kg/天，1次/天)或氨来占诺(等量于成人用药剂量150mg/天，1次/天)或等体积等浓度溶剂(模型)。同时设立空白对照，未感染小鼠口服给药等同于模型组。分组及各组动物给药剂量如下表所示：
  组别 药物 剂量 动物数
1 空白组(NC) 溶剂   18
2 模型组(M) 溶剂   18
3 3TC 拉米夫定 530mg/kg/天 10
4 ETV 恩替卡韦 0.03mg/kg/天 10
5 TDF 替诺福韦 530mg/kg/天 10
6 供试品低剂量组(A150) 氨来占诺 150mg/天* 10
*表示此处的剂量为换算成成人用药剂量后的值。换算方法如下：假设成人体重60kg，成人用药剂量/天＝小鼠用药剂量/kg/天*60kg/9.1。成人150mg/天等效于小鼠22.8mg/kg/天。
给药第5、7天时，各组小鼠尾静脉取适量的血，第9天小鼠处死后，取剩余外周血。所有血液样品离心取血清，ELISA检测HBV表面抗原(HBsAg)的浓度。具体方法参照ELISA试剂盒说明书。
结果如图17所示，可见，连续给150mg/天剂量氨来占诺5天后，血液中HBsAg水平显著降低，表明150mg/天剂量氨来占诺具有较好的抗乙肝活性。
实施例14氨来占诺在转基因鼠模型中的作用
雄性C57BL/6-HBV转基因小鼠(北京维通达生物技术有限公司提供)6周龄10只，随机分为2组(对照组5只，给药组5只)。各组动物给药剂量如下表：
Figure PCTCN2020098635-appb-000004
给药途径：灌胃，给药时使用1mL无菌注射器。
给药频率：供试品每天给药1次，连续给药35天，自由饮食饮水。
给药容量：10mL/kg，根据每只动物给药前最近一次体重，确定每只动物的给药量。
给药第7、14、21、35天时，各组小鼠尾静脉取适量的血，分离得到血清，用PBS分别稀释5000倍，用ELISA试剂盒检测HBV表面抗原(HBsAg)浓度。于酶标仪上测定450nm处的OD值。
抑制率按以下公式计算：
抑制率＝[(OD450对照孔-OD450给药孔)/OD450对照孔]×100％
采用HBV转基因小鼠模型对氨来占诺药效进行评价，HBV表面抗原HBsAg检测结果如图18所示，在给药浓度为48mg/kg条件下，氨来占诺能够抑制转基因小鼠体内HBsAg水平。
实施例15氨来占诺对小鼠体内免疫细胞的影响
6周龄雄性C57小鼠在短时间内通过尾静脉注射迅速推入病毒基因组DNA(基因型A)10μg/只，利用静脉高压模型建立病毒感染，并随机分组。注射后第二天起，根据化合物药时曲线，口服灌胃给药拉米夫定(3TC，530mg/kg/天，1次/天)、替诺福韦(TDF，530mg/kg/天，1次/天),恩替卡韦(ETV，0.03mg/kg/天，1次/天)，或指定浓度氨来占诺(4、8、16mg/kg/次，2次/天)或等体积等浓度溶剂(模型)。同时设立空白对照，未感染小鼠口服给药等同于模型组。分组及各组动物给药剂量如下表：
Figure PCTCN2020098635-appb-000005
第9天处死小鼠采集血液，裂红后用流式抗体染色。所有细胞在PBS中用抗 FcγRⅢ/Ⅱ(购自BD Biosciences)处理30分钟。CD3e-FITC(购自BD Biosciences)。CD4-PE、CD8a-APC-Vio770和CD34-FITC(GE)。CD19-APC、CD11b-PE-Cyanine7和CD117-efflor 450(购自eBioscience)。染色后样品采用500μL PBS混悬，通过BD AriaⅢ流式细胞仪分析。
结果如图19所示，氨来占诺对B细胞和T细胞均具有抑制作用，抑制作用具有剂量依赖性，此外，拉米夫定对免疫细胞也具有一定的抑制作用。随着浓度的增高，对免疫细胞的抑制增加，但对HBV的抑制作用反而增强，这也表明，氨来占诺不是通过免疫系统发挥抗HBV的作用。
实施例16细胞毒实验
以MTT法测试氨来占诺在HepG2,HCT-116,Hela,A549,MCF7,MKN45,THP-1,Huh7和293九种细胞内的毒性。将处于对数生长期的各种细胞，以6,000个/孔接种于96孔板，培养24h后，加入不同浓度氨来占诺(终浓度为100、50、25、12.5、6.25、3.125μM)，阳性对照盐酸阿霉素(终浓度为1μM)，空白对照组加等体积的培养液，溶剂对照组DMSO的用量以受试组所用的最大剂量0.1％为准，每个浓度设4个复孔。药物作用细胞72h后加入20μL 5mg/mL的MTT，37℃培养4h，加入100μL三联液(10％SDS、5％异丁醇、12mM盐酸)后，将96孔细胞培养板放置于37±1℃、5％CO2培养箱中过夜，于酶标仪上测定570nm处的OD值。
肿瘤细胞生长的抑制率按以下公式计算：
抑制率(％)＝[(OD570对照孔-OD570给药孔)/OD570对照孔]×100。
测试结果如表1所示，
表1
Figure PCTCN2020098635-appb-000006
可见，本发明的化合物在50μM浓度以下在测试的9种细胞内未见明显的细胞毒性。在100μM浓度下，在MKN45，HCT-116，MCF7和Huh7细胞内有一定细胞毒性，在其余5种细胞内未见明显的细胞毒性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 氨来占诺在制备抗病毒药物中的用途。
  2. 如权利要求1所述的用途，其特征在于，所述抗病毒药物为用于预防和/或治疗肝炎病毒感染的药物，或者是用于预防和/或治疗由于肝炎病毒感染所导致的疾病的药物。
  3. 如权利要求2所述的用途，其特征在于，所述肝炎病毒为乙肝病毒。
  4. 如权利要求2所述的用途，其特征在于，所述由于肝炎病毒感染所导致的疾病为急性肝炎、慢性肝炎、肝硬化、肝纤维化或肝癌。
  5. 如权利要求2所述的用途，其特征在于，所述由于肝炎病毒感染所导致的疾病为肝脏损伤。
  6. 一种药物组合物，其特征在于，包含：
    1)治疗有效剂量的氨来占诺，以及
    2)治疗有效剂量的另外一种或多种抗病毒活性物质，以及
    3)药学上可接受的载体。
  7. 如权利要求6所述的药物组合物，其特征在于，所述抗病毒活性物质为核苷或核苷酸类似物。
  8. 如权利要求7所述的药物组合物，其特征在于，所述核苷或核苷酸类似物为拉米夫定、齐多夫定、司坦夫定、替诺福韦、恩替卡韦、阿巴卡韦、扎西他滨、恩曲他滨、地丹诺辛、奈韦拉平、地拉韦啶、依非韦伦、依曲韦林、利匹韦林中的一种或多种。
  9. 如权利要求7所述的药物组合物，其特征在于，所述核苷或核苷酸类似物为拉米夫定、替诺福韦或恩替卡韦的中的一种或多种。
  10. 一种如权利要求6-9任一所述的药物组合物在制备用于预防和/或治疗肝炎病毒感染的药物，或者用于预防和/或治疗由于肝炎病毒感染所导致的疾病的药物中的用途。
  11. 如权利要求10所述的用途，其特征在于，所述肝炎病毒为乙型肝炎病毒。
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