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WO2008017264A1 - Composition pharmaceutique comprenant des donneurs de méthyle ou des promoteurs de donneur de méthyle et des composés antiviraux - Google Patents

Composition pharmaceutique comprenant des donneurs de méthyle ou des promoteurs de donneur de méthyle et des composés antiviraux Download PDF

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WO2008017264A1
WO2008017264A1 PCT/CN2007/070365 CN2007070365W WO2008017264A1 WO 2008017264 A1 WO2008017264 A1 WO 2008017264A1 CN 2007070365 W CN2007070365 W CN 2007070365W WO 2008017264 A1 WO2008017264 A1 WO 2008017264A1
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betaine
compound
treatment
methyl
group
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PCT/CN2007/070365
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Chinese (zh)
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Qingyan Zhou
Xiaoyuan Zhang
Yuanyou Li
Shangwu Wang
Xianfeng Ding
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Guangzhou Hezhu Biotechnology Co., Ltd.
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Publication of WO2008017264A1 publication Critical patent/WO2008017264A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • 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
    • A61K31/13—Amines
    • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/205—Amine addition salts of organic acids; Inner quaternary ammonium salts, e.g. betaine, carnitine
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4415—Pyridoxine, i.e. Vitamin B6
    • 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
    • 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
    • A61K31/515—Barbituric acids; Derivatives thereof, e.g. sodium pentobarbital
    • 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
    • A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7135—Compounds containing heavy metals
    • A61K31/714—Cobalamins, e.g. cyanocobalamin, i.e. vitamin B12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses

Definitions

  • the present invention relates to a novel use of a class of compounds which provide active methyl groups or participate in methyl transfer, and in particular to the use of such compounds as a medicament for the preparation of a medicament for the treatment of viral diseases.
  • Acute and chronic hepatitis caused by hepatitis G virus 8 kinds of human herpesvirus-induced retinitis, keratitis, interstitial pneumonia, encephalitis, genital herpes, herpes zoster and lip herpes; respiratory virus infection Bronchitis, pneumonia, measles, mumps and polio; acute gastroenteritis caused by enterovirus, diarrhea in travellers and infants; influenza A and B often cause seasonal global or local pandemics
  • the mortality rate of hemorrhagic fever with renal syndrome, Lhasa fever, Ebola hemorrhagic fever and dengue fever caused by various viruses is high.
  • Hepatitis C is also a common viral hepatitis, which is widespread worldwide. It is the leading cause of liver cirrhosis and liver cancer in countries such as Europe, the United States, and Japan. It is also the main cause of post-transfusion hepatitis in China.
  • the anti-HCV positive rate in our population is about 1% to 3%, so we estimate that there are about 30 million anti-HCV positive people in China.
  • Lamivudine and interferon are currently the most widely used antiviral drugs, mainly for the treatment of hepatitis B and hepatitis C.
  • the efficacy of interferon or lamivudine in combination with other drugs (including Chinese herbal medicine) in the treatment of chronic hepatitis B needs further confirmation.
  • Meta-analysis showed that HBeAg-positive patients were treated with common interferon alpha (general IFN alpha) for 4 to 6 months, and HBV-DNA negative rate in the treated and untreated groups (hybridization method) At 37% and 17%, respectively, the HBeAg negative rate was 33% and 12%, respectively, and the HBsAg negative rate was 7.8% and 1.8%, respectively.
  • Adefovir dipivoxil is an acyclic analog of 5'-monophosphate deoxyadenosine.
  • the standard treatment is polyglycolized a-interferon plus ribavirin, which is effective in only about half of the patients, and the other half of the failure is unclear. Therefore, finding a more effective treatment for hepatitis B and hepatitis C is a major difficulty in the field of liver disease research. For other viral diseases, similar problems exist and there is a need to find better treatments.
  • the object of the present invention is to provide a new use of a class of compounds which provide active methyl groups or participate in methyl transfer.
  • the use of such a compound as a medicine for treating a viral disease can significantly enhance the comprehensive antiviral effect of the body and the cells, and in particular, significantly enhance the inhibitory effect of known antiviral compounds on various viruses.
  • the compound of the present invention which can provide active methyl group or participate in methyl transfer is selected from the group consisting of folic acid and vitamin B12.
  • vitamin B6, vitamin B2, choline, betaine, serine and methionine are preferably used in combination with betaine or betaine, folic acid and vitamin B12.
  • the antiviral compound of the present invention is selected from one or a combination of one or more of an interferon compound and a nucleoside analog compound.
  • Nucleoside analog compounds include: abacavir, acyclovir, adefovir, amprenavir, atazanavir, delavirdine, didanosine, efavirenz, emtricita Bin, enfuvirtide, famciclovir, ganciclovir, indinavir, lamivudine, nelfinavir, nevirapine, oseltamivir, penciclovir, rimantadine, ritona Wei, Saquinavir, Stavudine, Tenofovir, Valvisicil, Zalunavir, Zidovudine, Sodium Phosphate and the like.
  • the viral diseases include various types of viral hepatitis, Japanese encephalitis, AIDS, influenza, and avian influenza.
  • the present invention also provides a pharmaceutical composition for treating a viral disease.
  • a pharmaceutical composition for treating a viral disease according to the present invention comprises: at least one compound which provides an active methyl group or participates in methyl transfer; and at least one antiviral compound.
  • the pharmaceutical composition is formulated into a dosage form selected from the group consisting of an injection, a powder injection, a capsule, a tablet, an oral solution, and a chewable tablet.
  • the clinical application route is mainly oral, but it can also be made into oral, sublingual, intranasal or various injections or rectal or inhaled or transdermal administration with various excipients.
  • Various dosage forms or ways to enter the human body are mainly oral, but it can also be made into oral, sublingual, intranasal or various injections or rectal or inhaled or transdermal administration with various excipients.
  • the present invention proves that the active methyl group or the compound involved in methyl transfer can enhance the antiviral ability of the body through various animal models and experiments, and can obviously enhance the comprehensive antiviral effect of the body and the cells, especially the known enhancement.
  • Compounds that provide reactive methylation or participate in methyltransfer are a general term for a class of elements related to intracellular methylation (Methylation), which generally include:
  • Folic acid, vitamin B12, vitamin B6, vitamin B2, choline, betaine, serine and methionine are all involved in the transfer of one carbon unit (methyl) in cells.
  • the main physiological function of active methyl group is as a source of methylation and donor.
  • the in vivo methylation process is to link these methyl small molecules with the corresponding substances under the action of related methylases in the body.
  • Corresponding biological functions, such as DNA methylation play a role in cell differentiation, gene regulation, inhibition of abnormal gene expression, and cancer formation. At least 37 methylases and about 100 substances in the body can be methylated to change their biological activities. Therefore, methylation has important physiological and biological significance in the human body.
  • Antiviral compounds refers to a class of compounds that have a therapeutic or prophylactic effect on viral diseases, including those under investigation, in bursts, and from commercially available compounds. Such compounds include, but are not limited to, interferon compounds, nucleoside analog compounds, and the like. Such a compound may be used in combination with one or more compounds in the present invention, or may be used sequentially.
  • Example 1 Effect of betaine combined with ⁇ -interferon and lamivudine on hepatitis B virus cell line cultured in vitro
  • 2.2 ⁇ 5 cell line is a foreign-established liver cancer cell line capable of actively secreting hepatitis B virus. It is derived from HepG2 liver cancer cells. It is known that ⁇ -interferon can inhibit 2.2.1.5 cell line through its intracellular signaling pathway. Hepatitis B virus is secreted. Lamivudine is a nucleoside analog that directly inhibits viral DNA replication. By measuring the levels of HBsAg, HBeAg and HBV-DNA in the supernatant of the culture solution, the antiviral effects of lamivudine and the biological activity of a-interferon can be identified. This example uses betaine, which provides one of active methyl groups or compounds involved in methyl transfer, in parallel with a-interferon and lamivudine in 2.2.1.5 cell lines to observe their inhibitory effect on hepatitis B virus.
  • MTT Feuka
  • HBsAg and HBeAg enzyme free kit ABOTT
  • ⁇ Select the appropriate concentration of ⁇ -interferon (200 IU / ml), lamivudine (200 ⁇ ⁇ / ⁇ 1) and the appropriate concentration of betaine (5 g / ml), respectively, in combination with the medium, A negative control group was set up.
  • HBsAg and HBeAg inhibition rate determination The cell supernatant stored at -20 °C was thawed on a 37 °C water bath, and the HBsAg and HBeAg titers were determined by the enzyme-free kit as a control, and were calculated by the following formulas.
  • Synergistic inhibition of drugs was performed using ⁇ -interferon (200 IU/ml), lamivudine (200 ⁇ ⁇ / ⁇ 1) and betaine (5 g/ml) as the compatibility combination, taking the 8th day Determination of the supernatant
  • V indicates that the corresponding drug is indicated; '-' indicates that the corresponding drug is not added.
  • Betaine at a concentration below 2 g/ml seems to promote the secretion of the virus, but there is no significant difference compared with the control. This may be related to the low concentration of betaine promoting cell growth. We hypothesize that betaine may inhibit the assembly and secretion of viral proteins by inhibiting endoplasmic reticulum stress, as the assembly of the virus primarily involves its own protein synthesis by inducing endoplasmic reticulum stress.
  • Lamivudine had no effect on cell proliferation inch less than 500 ⁇ ⁇ / ⁇ 1, when greater than 500 ⁇ ⁇ / ⁇ 1 inch display is significantly inhibited.
  • HCV protein indicates that the expression of HCV protein triggers the inhibition of STAT1 methylation and impairs the signaling process of Jak-STAT.
  • Unmethylated STAT1 is less active because it is inactivated by the inhibitor -STAT1 activation inhibitory protein (PIAS1).
  • SAM S-adenosylmethionine
  • betaine restored methylation of STAT1 and improved alpha-interferon signaling.
  • Betaine can significantly improve the antiviral effect of lamivudine, and their combined use of strontium is significantly greater than
  • the two effects of the two effects are simple and additive, and the mechanism may be related to the inhibition of intracellular virus assembly and secretion by betaine by inhibiting endoplasmic reticulum stress, and the betaine improves the activity of intracellular enzymes through protein methylation. Protein activity, through nucleotide methylation, promotes the conversion of intracellular dUTP to dTTP, enables cell replication to obtain sufficient thymidine, reduces cellular gene mutation and viral gene mutation, and enhances the comprehensive antiviral effect of cells.
  • Example 2 Betaine-enhanced lamivudine inhibits replication of hepatitis B virus in ducks
  • Duck hepatitis B is a hepatitis model of natural infection of ducks, which can lead to chronic hepatitis and cirrhosis. It is known that lamivudine is a nucleoside analogue that directly inhibits hepatitis B virus DNA replication in ducks. By detecting HBsAg in duck serum
  • the HBeAg and HBV-DNA content can be used to determine the antiviral activity of the drug.
  • betaine which is one of the compounds which provide active methyl group or participates in methyl transfer, is used in combination with lamivudine to observe their synergistic inhibitory effect on hepatitis B virus.
  • V indicates the addition of the corresponding drug
  • '-' indicates no corresponding drug
  • 1.6 PCR reaction Take 5 ⁇ 1 duck serum plus 50 ⁇ 1 lysate, 100. C was boiled for 10 min, centrifuged rapidly, placed on ice, and used as a template. For routine PCR reactions, positive sera were used as positive controls. A total of 5 positive controls were used for each reaction. The blank control contained all the components required for RT-PCR, but no template was added.
  • V indicates the addition of the corresponding drug
  • '-' indicates no corresponding drug
  • the DHBV titer (18 36.8+879.6) in duck serum was further decreased compared with the normal control group (2526.3 ⁇ 1022.6), and the antiviral effect was better than that of the lamivudine-only group.
  • Lamivudine and betaine treatment can significantly reduce the pathological changes of duck hepatitis B, the compatibility of the two
  • Example 3 Betaine combined with lamivudine inhibits the culture of Japanese encephalitis virus in vitro
  • Japanese encephalitis virus is a representative member of the Flaviviridae family, and a continuous infection model of Japanese encephalitis virus is established by using Japanese wild type encephalitis virus strain and human liver cancer cell line KN73. Through the virus inhibition experiment, the effect of betaine combined with lamivudine on the inhibition of Japanese encephalitis virus in vitro was observed.
  • the culture solution was changed until 90% of the cells showed cytopathic effect, and the remaining cells were further cultured, and cell passage was performed after formation of a monolayer.
  • Antiviral drugs for virus inhibition experiments Passage and culture of persistently infected cells KN73, after forming monolayer cells, add betaine 0-20 ⁇ ⁇ / ⁇ 1 and lamivudine without cytotoxicity 0-1 000 ⁇ ⁇ / ⁇ 1 ) The cells were cultured one day after administration and the cells were treated for 2 days. The amount of virus in the culture solution and the cells was measured by virus titration.
  • V indicates the addition of the corresponding drug
  • '-' indicates no corresponding drug
  • Virus titer determination method ⁇ hamster kidney cell plaque test method. The supernatants and cells of the virus-infected cells were collected and the cells were repeatedly frozen and thawed at -80 ° C and room temperature for 3 times, and the intracellular virus was released due to cell membrane rupture. Treated by double-resistance, respectively, 10-1, 10-2, 10-3, 10-4, 10-5...10
  • V indicates the addition of the corresponding drug
  • '-' indicates no corresponding drug
  • Betaine a compound that provides active methylation or participates in methyltransfer, slightly inhibits circulating Japanese encephalitis virus in cell culture and has synergistic inhibition with lamivudine.
  • Example 4 Betaine and ribavirin (ribavirin, ribavirin) combined application of HCV (C Effect of hepatic virus) RNA positive serum infection on HCV replication in human hepatoma cell line HepG2 cultured in vitro
  • Ribavirin monophosphate competitively inhibits inosine monophosphate dehydrogenase, reduces the synthesis of guanine nucleotides (GTP), and ribavirin triphosphate competitively inhibits GTP-dependent viral mRNA caps
  • GTP guanine nucleotides
  • ribavirin triphosphate competitively inhibits GTP-dependent viral mRNA caps The cap is formed to inhibit the replication of various RNA and DNA viruses.
  • HCV-RNA positive serum was infected with the human hepatoma cell line HepG2 cultured in vitro, and the effects of betaine and ribavirin on the replication of HCV in the cells were observed.
  • RT-PCR Reverse transcription polymerase chain reaction
  • PCR detection kit It is a product of Daan Gene Diagnosis Center of Sun Yat-Sen University.
  • RPMI1640 is a product of American Sigma
  • reverse transcriptase (MMLV) is a product of Promega, USA
  • TaqDNA polymerase and dNTP are products of Nippon Biotech.
  • Fluorescent quantitative PCR detection kit produced by Guangzhou Huayin Pharmaceutical Technology Co., Ltd.
  • HCV positive serum the final concentration of infected serum is 10%
  • Partial cells were collected on the 2nd, 4th, and 6th day after infection, and the culture supernatant was aspirated.
  • the 1640 solution was washed 5 times, 0.25% trypsin was digested to prepare a cell suspension, centrifuged at 1000 r/min for 5 min, the supernatant was discarded, and washed with PBS for 5 times, and then frozen at -20 ° C for testing.
  • RT-PCR detection of cultured cells HCV-RNA HC Use the HCV-RNA detection kit provided by Daan Gene Diagnosis Center of Sun Yat-sen University, and operate according to the instructions. Positive serum in the kit was used as a positive control. Subsequent experiments were performed after HCV-RNA was detected in the cultured cells.
  • [99] B Select the appropriate concentration of ribavirin (500 ⁇ ⁇ / ⁇ 1) and the appropriate concentration of betaine (5 g / ml), respectively, in combination with the medium, set the negative control group.
  • HCV-RNA titers in HepG cells See Table 9 for the effects of cell survival.
  • the results showed that ribavirin had an effective inhibitory effect on the secretion of viral antigens at a concentration above 10 g/ml, and had no obvious side effects on cell viability.
  • Betaine was in cells within lg/ml. Proliferation has a promoting effect, but the effect on viral RNA titer is not obvious. Therefore, we used ribavirin (500 ⁇ ⁇ / ⁇ 1) and betaine (5 g/ml) as a combination for synergistic inhibition test.
  • Ribavirin 500 ⁇ ⁇ / ⁇ 1
  • betaine 5 g/ml
  • V indicates the addition of the corresponding drug
  • '-' indicates no corresponding drug
  • ribavirin has no obvious inhibitory effect on cell viability within 100 ( ⁇ g/ml), while betaine has a slight promoting effect on cell growth within 1 g/ml, to l ( ⁇ g /ml showed a slight boost.
  • 3.2 ribavirin showed an inhibitory effect on intracellular hepatitis C virus at 10 ( ⁇ g/ml ⁇ , IC 5Q was: 777.3 ⁇ ⁇ / ⁇ 1 ⁇ and betaine was within 1 ( ⁇ g/ml for intracellular) Hepatitis C virus did not show significant inhibition.
  • Ribavirin 50 ( ⁇ g / ml) and betaine (5 g / ml) as a combination, showing a significant synergistic inhibition of the amount of viral RNA.
  • betaine can restore the methylation of STAT1 in cells and improve the signal transduction of ⁇ -interferon.
  • Example 5 Clinical trial of betaine and lamivudine in the treatment of chronic hepatitis B
  • test cases For patients with positive chronic hepatitis B, the test cases must meet the following conditions:
  • ALT continues or rises repeatedly; 6. Hepatic histology has hepatitis lesions.
  • Chronic hepatitis B treatment mainly includes antiviral, immune regulation, anti-inflammatory and liver protection, anti-fibrosis and symptomatic treatment.
  • Treatment group Compounds that provide active methylation or participate in methyl transfer, 0.3g each time, 3 times a day, and conventional lamivudine treatment, the first course of treatment is 12 months. Some cases are necessary to continue the second treatment Cheng, the dose is increased by 1-4 times according to the first course of treatment, and the daytime is 12 months.
  • Control group Regular lamivudine treatment, 12 months for each course of treatment, 1-2 courses can be observed.
  • Symptoms fatigue, anorexia, pain, bloating, nausea, vomiting, low fever, joint pain, bleeding gums, nasal discharge, etc.
  • Pre-treatment symptoms are: no, ten, - indicates that after treatment, the symptoms disappear, reduce, remain unchanged, aggravate with '-, earth,
  • +, ++' indicates. Observe and record once before treatment, every week during treatment, at the end of treatment, and 3 months after the end of treatment. The severity of clinical symptoms is 0-3, and the criteria are as follows: 0
  • Points asymptomatic; 1 point, mild, slightly noticeable symptoms; 2 points, moderate, conscious symptoms but not affecting work; 3 points, significantly affecting work and life.
  • sALT serum alanine aminotransferase
  • sAST serum aspartate aminotransferase
  • AFP alpha-fetoprotein
  • serum total protein albumin, and globulin were tested before treatment, monthly during treatment, at the end of treatment, and 3 months after treatment.
  • HBV serological markers including HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HB
  • -HBc and anti-HBc-IgM were tested once before treatment, every month during treatment, and at 3, 6, 9, and 12 months after treatment.
  • HBeAg negative conversion rate HBV-DNA ⁇ 10 5 copies / ml and ALT recurrence.
  • Virological response refers to serum HBV-DNA not detected (PCR method) or lower than the lower limit of detection, or Baseline drop ⁇
  • Serological response Refers to serum HBeAg negative or HBeAg seroconversion or HBsAg negative or HBsAg seroconversion.
  • Biochemical response Refers to serum ALT and AST return to normal.
  • ALT is normal. 5.
  • Rebound The initial response was reached, but HBV-DNA levels were re-raised without change of treatment, or turned positive after one degree of negative conversion, with or without ALT elevation. ⁇ also refers to ALT and AST after repeated, no change in treatment, but should be ruled out by other factors caused by elevated ALT and AST. 6.
  • Recurrence The response to the end of treatment is reached, but HBV-DNA is re-raised or positive after stopping the drug. Astringent also means that ALT and AST are re-elevated after stopping the drug, but ALT caused by other factors should be excluded. And AST increased.
  • Treatment group Compound capsules that can provide active methyl group or participate in methyl transfer 0.3g/time
  • the 9600 Fluorescence DNA Analyzer is manufactured by Biotronics, USA.
  • Table 12 Effects of compounds that provide active methylation or participate in methyltransfer on pathogenic indicators in patients with hepatitis B
  • Compounds that provide active methylation or participate in methyltransfer can accelerate the recovery of hepatocyte function by protecting hepatocytes and significantly promoting hepatocyte albumin synthesis.
  • Example 6 Clinical trial of betaine alone, betaine and interferon in the treatment of chronic hepatitis B
  • Treatment group 1) Betaine group alone: Compound capsules that can provide active methyl group or participate in methyl transfer 0.6g/time, three times a day orally; 24 weeks for one course of treatment. 2) Interferon combined with betaine group: Compound capsules that can provide active methyl group or participate in methyl transfer 0.6g/time, three times a day orally; Interferon 5 ⁇ 10MU, 1/d or 3/week, 24 weeks for a course of treatment . 3) Interferon group: Interferon alone 5 ⁇ 10MU, 1/d or 3/week, the same treatment as before. 4) Negative control group, routine liver protection treatment, 24 weeks for a course of treatment.
  • Serum ALT is according to the Lai's method.
  • Quantitative analysis of HBsAg and HBeAg The automatic fast-acting micro-enzyme-free analytical system is used, and the instruments and reagents are all products of Abbott.
  • Quantitative detection of HBV-DNA ⁇ Quantitative polymerase chain reaction (PCR) detection, AG-9600 fluorescent DNA analysis detector manufactured by Biotronics, USA.
  • betaine alone has a certain therapeutic effect on hepatitis B virus.
  • influenza virus, AIDS, and avian influenza virus can also be treated with an antiviral compound such as an interferon compound or a nucleoside analog compound of the present invention, it is also obtained by experiments similar to those of Examples 1 to 6.
  • an antiviral compound such as an interferon compound or a nucleoside analog compound of the present invention
  • Example 7 Using betaine as the main ingredient to prepare an injection
  • Example 8 Making a powder injection using betaine as a main material
  • the concentration was adjusted to pH 7.0 with 1 mol/L of HCl, and mannitol was added to make the final concentration of mannitol 4%, added to the bottle in an amount of 2-20 ml (preferably 2 ml), and lyophilized for use.
  • Example 9 Making a capsule by using betaine as a main material
  • Example 10 Making a tablet using betaine as a main material
  • Example 11 Oral solution made from betaine
  • Example 12 Making chewable tablets using betaine as main material
  • Example 13 using betaine as the main material, with multivitamins and adjuvants, according to the methods of Examples 9 to 12, can be made into oral liquid, capsules, or oral tablets or chewable tablets, can also be made Food or health products.
  • VE is 100 parts
  • VA is 5 parts
  • VC is 50 parts
  • 50 parts of nicotinic acid 0.3 parts of biotin
  • 1 part of pantothenic acid folic acid
  • Example 14 using betaine, folic acid and vitamin B12 as the main material, with multivitamins and auxiliary materials can be made into oral liquid, capsule, or oral tablets or chewed by the methods of Examples 9 to 12. Tablets can also be made into food or health products.
  • 'VB2 is 20 parts, VB6 is 50 parts, VE is 100 parts, VA is 5 parts, VC is 50 parts, nicotinic acid is 50 parts, biotin is 0.3 parts, pantothenic acid is 1 part; trace elements are appropriate.
  • Example 15 The use of betaine as the main material with antiviral drugs (nucleoside analogues, such as lamivudine) and adjuvants into capsules
  • the capsule size and capacity are not limited.
  • Example 16 Use of betaine as a main material with antiviral drugs (nucleoside analogues such as lamivudine) and adjuvants to make tablets
  • Example 17 Using betaine, folic acid and vitamin B12 as the main material, together with antiviral drugs (nucleosides such as lamivudine) and auxiliary materials to make capsules
  • the final amount of betaine containing capsules is O.lg / grain or 0.3g / grain
  • the lamivage is 0.02 g/grain or 0.06 g/grain. This formula does not limit the size and volume of the capsule.
  • Example 18 Using betaine, folic acid and vitamin B12 as the main ingredient, combined with antiviral drugs (nucleosides such as lamivudine) and adjuvants to make tablets
  • Example 19 Using betaine as the main material, with interferon as an injection
  • the pH of HC1 was adjusted to 7.0 to make a 2-20 mr injection. A 2 ml injection is preferred.
  • Example 20 Using betaine as the main material, with interferon as a powder injection
  • the pH of HC1 was adjusted to 7.0, and mannitol was added to make the final concentration of mannitol 4%, and the amount was added to the bottle in an amount of 2-20 ml (preferably 2 ml), and lyophilized for use.

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Abstract

L'invention concerne une composition pharmaceutique pour le traitement de maladies virales, comprenant : a) au moins un donneur de méthyle ou un promoteur de donneur de méthyle, de préférence de la bétaïne ou une combinaison de bétaïne, d'acide folique et de vitamine B12, et 2) (au moins un composé antiviral), de préférence de l'interféron ou de la lamivudine. Des donneurs de méthyle et des promoteurs de donneur de méthyle augmentent l'effet antiviral et l'effet thérapeutique de composés antiviraux pour traiter des maladies virales.
PCT/CN2007/070365 2006-08-01 2007-07-26 Composition pharmaceutique comprenant des donneurs de méthyle ou des promoteurs de donneur de méthyle et des composés antiviraux WO2008017264A1 (fr)

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CN104174013B (zh) * 2014-08-16 2015-07-08 广州一品红制药有限公司 一种含促肝细胞生长素的组合物和应用
CN106036823A (zh) * 2016-05-23 2016-10-26 沈阳迪格医疗科技有限公司 一种保健品及其制备方法
CN106509577A (zh) * 2016-10-31 2017-03-22 江西熙帝生物科技有限公司 一种具有护肝功能的饮料冲调颗粒

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EP4011371A4 (fr) * 2019-08-06 2024-02-07 Lianyungang Jinkang Hexin Pharmaceutical Co. Ltd. Composition pharmaceutique produisant une quantité sûre de monoxyde d'azote dans le corps et utilisation associée

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