CN106619591B - The purposes and pharmaceutical composition of oxetacaine in medicine preparation - Google Patents
The purposes and pharmaceutical composition of oxetacaine in medicine preparation Download PDFInfo
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Abstract
The invention proposes oxetacaine or its pharmaceutically acceptable salt purposes in medicine preparation and a kind of for treating or preventing hepatitis b virus infected pharmaceutical composition.The assembling of hepatitis B virus particles can be effectively suppressed in the drug or pharmaceutical composition, can be effectively used for the infection for treating or preventing hepatitis type B virus.
Description
Technical field
The present invention relates to biomedicine field, in particular it relates to oxetacaine purposes in medicine preparation and
Pharmaceutical composition, more particularly it relates to the purposes of oxetacaine or its pharmaceutically acceptable salt in medicine preparation
And it is a kind of for treating or preventing hepatitis b virus infected pharmaceutical composition.
Background technique
Hepatitis type B virus (Hepatitis B Virus, HBV) infection is that slow virus the most universal in the world is sexy
Dye.HBV mainly passes through the propagation of the approach such as blood born, sexual behaviour propagation, mother-to-baby transmission.Hepatitis B virus infection is that the world is top
Health problem leads to have 768000 people dead every year in the whole world, is the tenth-largest cause of death in the world.
And treat the medicinal application of hepatitis B virus infection at present only there are two types of interferon in clinical, it is divided into interferon
(IFN) and two kinds of forms of Peg-IFN alpha-2b (PEG-IFN), successively ratified in 1990 and 2005 by FDA；With five kinds of nucleosides
The viral polymerase inhibitors drug of analog, i.e. Lamivudine (Lamivudine, 3TC or LMV), adefovirdipivoxil
(Adefovir, ADV), Entecavir (Entecavir, ETV), Sebivo (Telbivudine, LdT) and tenofovir
(Tenofovir disoproxil fumarate,TDF).But interferon is only effective to the patient less than 40%, and also
Stronger side effect；And polymerase inhibitors cannot remove cccDNA, the duplication of drug withdrawal restrovirus can quick rebound, and take for a long time
Can also virus be made to morph and develop drug resistance.
Therefore, the drug for screening new anti-hepatitis B virus infective shows especially urgent and important.
Oxetacaine (Oxethazaine) is clinically always to use as an effective local anesthetic, simultaneously
Oxetacaine can also be controlled by a variety of gastrointestinal disorders, such as esophagitis, pain caused by chronic gastritis and peptic ulcer and
The secretion of gastric acid inhibitory.
However, the purposes of oxetacaine need further to develop.
Summary of the invention
The present invention is directed to solve at least some of the technical problems in related technologies.
The application is discovery and the understanding based on inventor to following facts and makes:
Oxetacaine can obviously inhibit the secretion of HBeAg (the e antigen of secreting type), and total to HBV virus in liver cell
DNA has apparent inhibiting effect, meanwhile, oxetacaine can also significantly inhibit the assembling of HBV virion.This illustrates former times difficult to understand
Cacaine is to be assembled into target with virion, influences the assembling of the virion in liver cell, and then inhibit virus in the cell
Duplication.
Target, resistance of hepatitis B are assembled into HBV virion in preparation for this purpose, the invention proposes oxetacaines
New application in the drug of virus infection.
In the first aspect of the present invention, the invention proposes oxetacaines or its pharmaceutically acceptable salt to prepare drug
In purposes.According to an embodiment of the invention, the drug is for inhibiting hepatitis B virus particles to assemble；Or treatment or pre-
Prevent hepatitis b virus infected.According to an embodiment of the invention, oxetacaine or pharmaceutically acceptable salt can significantly inhibit HBV
Nucleocapsid assembling and liver cell in HBV virus total DNA, therefore, oxetacaine proposed by the invention or its can pharmaceutically connect
The purposes of the salt received in medicine preparation, the drug can significantly inhibit hepatitis B virus particles assembling；Or significant treatment or
Person's prevention is hepatitis b virus infected.
According to an embodiment of the invention, the purposes of above-mentioned oxetacaine or its pharmaceutically acceptable salt in medicine preparation
It can further include at least one following additional technical feature:
According to an embodiment of the invention, the drug is for treating or preventing human hepatitis B virus infection.At this
In some embodiments of invention, inventors have found that can to significantly inhibit human liver cancer thin for oxetacaine or its pharmaceutically acceptable salt
The nucleocapsid assembling of HBV and cell conditioned medium and intracellular HBV virus total DNA in born of the same parents system HepAD38 cell.Therefore, institute of the present invention
The purposes of the oxetacaine of proposition or its pharmaceutically acceptable salt in medicine preparation, the drug is for treating or preventing
The effect of human hepatitis B virus infection further increases.
In the second aspect of the present invention, it is hepatitis b virus infected for treating or preventing that the invention proposes a kind of
Pharmaceutical composition.According to an embodiment of the invention, described pharmaceutical composition includes: oxetacaine or its pharmaceutically acceptable salt
As active constituent.According to an embodiment of the invention, oxetacaine significantly inhibits HBV in the nucleocapsid assembling and liver cell of HBV
Viral total DNA can be significantly inhibited using oxetacaine or its pharmaceutically acceptable salt as the pharmaceutical composition of active constituent
HBV virion is assembled into target spot, hepatitis b virus infected effective for treating or preventing.
According to an embodiment of the invention, aforementioned pharmaceutical compositions can further include pharmaceutically acceptable carrier.
According to an embodiment of the invention, the stability and effective component of described pharmaceutical composition can be improved in pharmaceutically acceptable carrier
Using degree, to further improve described pharmaceutical composition to hepatitis b virus infected treatment or prevention.
According to an embodiment of the invention, described pharmaceutical composition further comprises second medicament, the second medicament is different
In oxetacaine or its pharmaceutically acceptable salt and for treating or preventing hepatitis b virus infected.According to the present invention
Embodiment, the second medicament and oxetacaine or the combination of its pharmaceutically acceptable salt further improve the medicine group
Object is closed to hepatitis b virus infected treatment or prevention.
According to an embodiment of the invention, the second medicament includes selected from following at least one: interferon (IFN) gathers
Ethylene glycol interferon (PEG-IFN), Lamivudine (Lamivudine, 3TC or LMV), adefovirdipivoxil (Adefovir, ADV), grace
For card Wei (Entecavir, ETV), Sebivo (Telbivudine, LdT), tenofovir (Tenofovir disoproxil
fumarate,TDF).Lamivudine, adefovirdipivoxil, Entecavir, Sebivo, tenofovir are that HBV varial polymerases inhibit
Agent drug.The target spot of the inhibition HBV virus infection of oxetacaine or its pharmaceutically acceptable salt is different from above-mentioned second medicament,
Oxetacaine or its pharmaceutically acceptable salt are assembled into target spot with nucleocapsid, and there is no so that virus is developed drug resistance the wind of mutation
Danger can continuously and effectively achieve the purpose that remove hepatitis type B virus.According to an embodiment of the invention, above-mentioned second medicament is extremely
It is one of few to be combined with oxetacaine or its pharmaceutically acceptable salt, described pharmaceutical composition to hepatitis b virus infected
Treatment or preventive effect are more significant.
According to an embodiment of the invention, described pharmaceutical composition in tablet, injection, pulvis, elixir, capsule, suspension,
Syrup, pill, sustained release preparation, controlled release preparation or nanometer formulation.The pharmaceutical composition of various dosage forms is to be expected to prescription
Formula administration, inhibits the assembling of HBV virion, to further effectively treat or prevent hepatitis b virus infected.
According to an embodiment of the invention, described pharmaceutical composition is for treating or preventing human hepatitis B virus sense
Dye.In some embodiments of the invention, inventors have found that oxetacaine can to significantly inhibit Bel7402 HepAD38 thin
In born of the same parents HBV nucleocapsid assembling and cell conditioned medium and intracellular HBV virus total DNA, described pharmaceutical composition for treat or
It is more significant to prevent human hepatitis B virus infectious effect.
According to some embodiments of the present invention, using oxetacaine or its pharmaceutically acceptable salt as the drug of active constituent
Composition may include pharmaceutically acceptable carrier, and the dosage form of pharmaceutical composition and administration mode are not particularly limited.It is right
In oral administration, which may include adhesive, lubricant, disintegrating agent, excipient, solubilizer, point
Powder, stabilizer, suspending agent, colorant and aromatic.For ejection preparation, pharmaceutically acceptable carrier may include buffering
Agent, preservative, analgesic, solubilizer, isoosmotic pressure agent (isotonic agent) and stabilizer.For the preparation of local administration,
Pharmaceutically acceptable carrier may include alkali, excipient, lubricant and preservative.Pharmaceutical composition of the invention can with it is upper
The pharmaceutically acceptable carrier stated combines and is prepared to various dosage forms.For example, for oral administration, pharmaceutical composition can be by
It is prepared into small pieces, tablet, capsule, elixir, suspension or syrup.For ejection preparation, pharmaceutical composition can be prepared to example
Such as the ampoule of the dosage form of dose or the haplotype dosage form of such as multi-dose container.Pharmaceutical composition can also be prepared to molten
Liquid, suspension, tablet, pill, capsule, durative action preparation, sustained release preparation, controlled release preparation or nanometer formulation.
Wherein, some specific examples according to the present invention, be suitble to excipient and dilution in the carrier of pharmaceutical formulation can
To include: lactose, glucose, sucrose, D-sorbite, mannitol, xylitol, erythritol, maltitol, starch, Arab
Rubber, alginates, gel, calcium phosphate, calcium silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone,
Water, methyl hydroxybenzoate, Nipasol, talcum, magnesium stearate and mineral oil.
Other embodiments according to the present invention can also include filler, anticoagulation in pharmaceutical composition of the invention
Agent, lubricant, moisturizer, aromatic and preservative.
According to an embodiment of the invention, of the invention using oxetacaine or its pharmaceutically acceptable salt as the medicine of active constituent
Compositions can inhibit the assembling of hepatitis B virus particles, significantly under the concentration of no cytotoxicity so as to control
It treats or is administered when preventing hepatitis b virus infected.
Term " administration " used in herein, which refers to, introduces patient by certain suitable mode for the substance of predetermined amount.
Drug or pharmaceutical composition of the invention can be administered by any common approach, as long as it can reach expected group
It knits.The various modes of administration are expected, including peritonaeum, vein, muscle, and subcutaneously, cortex takes orally, part, nasal cavity, lung
Portion and rectum, but the administration mode that the present invention is not restricted to these has illustrated.However, when due to oral administration, oral administration
The active constituent of composition should be coated or be formulated to that it is prevented to be degraded in stomach.Preferably, composition of the invention
It can be administered with ejection preparation.In addition, the spy that active constituent is transmitted to target cell can be used in pharmaceutical composition of the invention
Instrument is determined to be administered.
The administration frequency and dosage of pharmaceutical composition of the present invention can be determined by multiple correlative factors, which includes
The disease type to be treated, administration route, patient age, gender, weight and the severity of disease and as activity at
The drug type divided.According to some embodiments of the present invention, daily dose can be divided into 1 dose, 2 doses or multi-agent of suitable form, with
With 1 time, 2 times or multiple dosing in the entire period, as long as reaching therapeutically effective amount.
Term " therapeutically effective amount " refers to that compound is enough to significantly improve the amount of certain symptoms relevant to disease or illness,
It also is that given illness and dosage regimen provide the amount of therapeutic effect.For example, reduced in treating hepatitis B virus infection,
The drug or compound for preventing, delay, inhibiting or blocking any symptom of disease or illness should be that treatment is effective.Treatment has
The pharmaceutical composition or compound of effect amount do not need to cure disease or illness, but will provide treatment for disease or illness, so that a
The symptom that the breaking-out of the disease or illness of body was delayed, and prevented or prevented perhaps disease or illness alleviated or disease or
The time limit of illness is changed such as disease or illness become not serious, or accelerates rehabilitation.
Term " treatment " obtains desired pharmacology and/or physiologic effect for referring to.The effect is with regard to complete or partial
It can be preventative for prevention disease or its symptom, and/or just partially or completely cure caused by disease and/or disease not
It can be for good action therapeutic." treatment " used herein covers mammal, particularly the disease of people (refers mainly to second
Hepatitis virus infection) treatment, comprising: (a) prevent in being easy the individual that illness but not yet make a definite diagnosis is fallen ill disease (such as
Prevent hepatitis b virus infected) or illness generation；(b) inhibit disease, such as retardance disease development；Or (c) alleviate disease, example
As mitigated symptom relevant to disease." treatment " used herein cover by drug or compound give individual to treat, cure,
Any medication of the disease alleviating, improve, mitigating or inhibit individual, will including but not limited to contain it is described herein with oxetacaine or
Its pharmaceutically acceptable salt is that the pharmaceutical composition of active constituent gives individual in need.
According to an embodiment of the invention, oxetacaine drug of the invention or pharmaceutical composition can be with conventional treatments
And/or therapy is used in combination, or can be used separately with conventional treatments and/or therapy.When oxetacaine of the invention
When drug or pharmaceutical composition are administered in using the conjoint therapy with other medicines, they can sequentially or simultaneously give a
Body.Alternatively, pharmaceutical composition of the invention may include oxetacaine of the invention, pharmaceutically acceptable carrier or pharmaceutically may be used
The combination of the excipient of receiving and other medicines known in the art or preventive medicine.
According to an embodiment of the invention, for human body, it is lasting to take medicine under the therapeutic dose of 0.2-0.4mg/kg oxetacaine
Discovery does not cause toxic reaction, the oral oxetacaine for launching 20mg of the adult of 50kg, the former times card difficult to understand in blood plasma after one hour
The maximum concentration of cause is about 20 μ g/mL (about 40 μM), much higher than the effective concentration of oxetacaine, the bioavilability of oxetacaine
Height, small toxicity, metabolic pathway are clear.
According to an embodiment of the invention, the acceptable salt form of oxetacaine of the present invention is all useful.Term is " pharmaceutically
Acceptable salt " refers to that those salt forms are it will be apparent that i.e. their substantially nontoxic and energy for pharmaceutical chemistry man
Pharmacokinetic property, palatability, absorption, distribution, metabolism or excretion needed for providing.
Detailed description of the invention
Fig. 1 is according to embodiments of the present invention 1 oxetacaine (Oxethazaine) Anti-HBV effect testing result figure,
Wherein, Figure 1A is the structure chart of oxetacaine,
Figure 1B is that oxetacaine handles 3 days cytotoxicities to HepAD38 cell,
Fig. 1 C be oxetacaine can it is dose-dependent inhibit cell conditioned medium in HBeAg result figure,
Fig. 1 D be oxetacaine can it is dose-dependent inhibit cell conditioned medium HBV total DNA real-time quantitative PCR as a result,
Fig. 1 E be oxetacaine can the dose-dependent real-time quantitative PCR for inhibiting intracellular HBV total DNA as a result,
Fig. 1 F is that oxetacaine can the dose-dependent Southern blot result for inhibiting intracellular HBV total DNA
Figure；
Fig. 2 is the result that according to embodiments of the present invention 2 oxetacaine does not influence the protein expression under CMV promoter control
Figure；
Fig. 3 is the knot that according to embodiments of the present invention 2 oxetacaine can effectively lower the content of HBV total DNA intracellular
Fruit figure,
Wherein, Fig. 3 A shows that oxetacaine handles 6 days cytotoxicities to HepAD38 cell,
Fig. 3 B shows influence of the oxetacaine to cccDNA intracellular,
Fig. 3 C shows influence of the oxetacaine to HBV total DNA intracellular；
Fig. 4 be according to embodiments of the present invention 2 oxetacaine for HBV precore mRNA, pgRNA and HBV total serum IgE
The result figure that has no significant effect of content；
Fig. 5 be according to embodiments of the present invention 2 oxetacaine to the HBV DNA in the assembling of the nucleocapsid of HBV and nucleocapsid
There is the result figure of significant dose-dependent inhibitory effect；And
Fig. 6 is according to embodiments of the present invention 3 antiviral effect to nucleoside analog class Drug Resistance HBV mutant strain
Result figure.
Specific embodiment
Below in conjunction with specific embodiment detailed description of the present invention embodiment, the following examples are merely to illustrate this hair
It is bright, and should not be taken as limiting the scope of the invention.Particular technique or condition are not specified in embodiment, according to text in the art
Offer described technology or conditions (such as with reference to J. Pehanorm Brooker etc. write, " Molecular Cloning:A Laboratory guide " that Huang Peitang etc. is translated,
The third edition, Science Press) or carry out according to product description.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
The evaluation of 1 oxetacaine Anti-HBV effect of embodiment
1. experimental material
1.1 cells and drug
HepAD38 cell line: human liver cancer stably transfected cell line is integrated with HBV gene group in chromosome, can support
HBV DNA synthesis and protein expression, can produce infectious virus particle.Viral duplication is by tetracycline in this cell line
The CMV promoter control of regulation.
Oxetacaine is purchased from Sigma company, and Lamivudine (3TC) is NIH present.
1.2 reagent
DMEM/F12 culture medium and fetal calf serum (FBS) are purchased from GIBCO company；HBV surface antigen (S antigen) and e antigen
Detection kit is purchased from Shanghai Ke Hua Biotechnology Co., Ltd；Reagent is purchased from Invitrogen company；iTaq
Universal SYBR Green supermix reagent is purchased from Bio-rad company；DIG High Prime DNA Labeling
And Detection Starter Kit II is purchased from Roche company.
2. experimental method and result
The cytotoxicity of 2.1 oxetacaines (Oxethazaine) detects
1) HepAD38 cell is inoculated in 96 porocyte culture plates by 8000 cells/wells (100 μ L), 37 DEG C of cell trainings
It supports and is cultivated in case, it is spare after cell is adherent.
2) agent-feeding treatment cell: use cell culture fluid (DMEM/F12+10% fetal calf serum) by drug with 2 times of gradient dilutions
6-10 gradient, 3 multiple holes of every gradient.The 100 μ L cell culture fluid for containing drug is added in the cell in step (1),
Continue in 37 DEG C of cell incubators cultivate 72h after in case detection.
3) cell culture supernatant containing drug is discarded, every hole, which is added, contains 10%The culture solution of reagent
100 μ L, after being incubated for 1h-4h in 37 DEG C of incubators, with light absorption value at all band microplate reader detection 540nm wavelength, tuning wavelength is
620nm, and each concentrations of cells survival rate is calculated, or detection fluorescent value (excitation wavelength: 540nm.Wavelength of transmitted light: 595nm)
To calculate cell survival rate.
As a result as shown in Figure 1B.
Figure 1B is as the result is shown: oxetacaine handles 3 days CC to HepAD38 cell50(half cytotoxic concentration) is
54.7 micromoles per liters.
The detection of 2.2 oxetacaines (Oxethazaine) Anti-HBV effect
1) HepAD38 cell is inoculated in 96 porocyte culture plates by 8000 cells/wells (100 μ L), 37 DEG C of cell trainings
It supports and is cultivated in case, it is spare after cell is adherent.
2) agent-feeding treatment cell: with cell culture fluid (DMEM/F12+10% fetal calf serum) that drug is dilute with 2 times of gradients
It releases, 100 microlitres of cell culture fluids containing drug are added in the cell in step (1) by 3 multiple holes of every gradient, 37 DEG C thin
Continue to cultivate 72h in born of the same parents' incubator in case detection.
3) cell conditioned medium and cell are collected respectively, with the HBeAg (secretion in ELISA detection kit detection cell conditioned medium
Type e antigen) and HBsAg (surface antigen) content.
4) in addition distinguish the cell conditioned medium and intracellular HBV total DNA collected in extraction step (3), use real-time quantitative PCR
Method detection cell conditioned medium and intracellular HBV total DNA.
Real-time quantitative PCR the primer is as shown in NO:1~4 SEQ ID:
Primer (HBV S144- forward primer): 5 '-TCACCAACCTCTTGTCCT-3 ' (SEQ ID NO:1).
Primer (HBV S144- reverse primer): 5 '-GACAAACGGGCAACATACCT-3 ' (SEQ ID NO:2).
Primer (GAPDH forward primer): 5 '-GAAGGTGAAGGTCGGAGTC-3 ' (SEQ ID NO:3).
Primer (GAPDH reverse primer): 5 '-GAAGATGGTGATGGGATTTC-3 ' (SEQ ID NO:4).
Meanwhile intracellular HBV total DNA is extracted, intracellular HBV total DNA is detected with the method for Southern blot,
Experimental procedure is as follows:
1) by HepAD38 cell 2 × 105Cells/well is inoculated in 6 porocyte culture plates, is trained in 37 DEG C of cell incubators
It supports, it is spare after cell is adherent.
2) agent-feeding treatment cell: with cell culture fluid (DMEM/F12+10% fetal calf serum) that drug is dilute with 2 times of gradients
It releases.Culture medium containing drug is added in cell, continue cultivate cell 72h after in case detection.
3) cell is collected, lytic cell simultaneously extracts intracellular HBV total DNA, is detected with the method for Southern blot thin
HBV total DNA intracellular.
As a result as shown in Fig. 1 C~1F.Wherein, the 3TC in figure indicates that Lamivudine pharmaceutical group is added, and C is blank control
Group, i.e., non-dosing group, RC indicate that the cyclic annular dsdna segment of relaxation, SS indicate single stranded DNA.As the result is shown: oxetacaine can agent
The HBeAg, EC inhibited in cell conditioned medium that amount relies on50(half effective concentration) is about 10 micromoles per liters, and to HBsAg without
It acts on (as shown in Figure 1 C)；The oxetacaine as the result is shown of real-time quantitative PCR can dose-dependent inhibition cell conditioned medium (such as figure
Shown in 1D) and intracellular (as referring to figure 1E) HBV total DNA, EC50Respectively 1.25 micromoles per liters and 2.5 micromoles per liters,
This result shows that oxetacaine be for the inhibiting rate of HBV total DNA intracellular and extracellular it is comparable, also just eliminating is former times difficult to understand
Cacaine inhibits a possibility that HBV viral secretory；The result of Southern blot analysis also shows oxetacaine can be with dose-dependant
The intracellular HBV total DNA (as shown in fig. 1F) of inhibition, oxetacaine is for mature genomic DNA rcDNA and genome
The intermediate product ssDNA of duplication has the apparent and comparable inhibition of degree.This result also indicates that oxetacaine processing not
There is the ratio for substantially changeing rcRNA/ssDNA, does not also directly affect the DNA synthesis of HBV.This result shows that oxetacaine very
Antiviral effect may be realized by the virus replication phase before influencing viral DNA synthesis.The result of Fig. 1 also indicates that Austria
Former times cacaine does not influence the DNA synthesis and its later virus replication phase of virus.Therefore inventor infers that oxetacaine may shadow
Ring the synthesis and degradation of the cccDNA (closed hoop DNA) in the HBV history of life, the transcription of HBV RNA, the assembling of HBV nucleocapsid
Stage.
2 oxetacaine of embodiment (Oxethazaine) directly acts on the assembling of HBV nucleocapsid and plays antivirus action
1. experimental material
1.1 cells, plasmid and drug
HepAD38 cell line: human liver cancer stably transfected cell line is integrated with HBV gene group in chromosome, can support
HBV DNA synthesis, protein expression can produce infectious virus particle.Viral duplication is tetracycline tune in this cell line
The CMV promoter control of control.
Huh7 is source of people liver cancer cells, is given for Wuhan institute of viruses Chen Xinwen researcher.
PFlag-GAPDH is the GAPDH egg of the expression fusion Flag label under the CMV promoter control of this laboratory building
White plasmid；
Oxetacaine is purchased from Sigma company, and Lamivudine (3TC) is NIH present.
1.2 reagent
MEM and DMEM/F12 culture medium and fetal calf serum (FBS) are purchased from GIBCO company；Reagent and transfection
Reagent Lipofectamine 2000 is purchased from Invitrogen company；iTaq Universal SYBR Green supermix
Reagent is purchased from Bio-rad company；The rabbit source of Anti-HBV activity core albumen is mostly anti-, the mouse of the source of mouse monoclonal antibody of anti-flag and anti-β-actin
Source monoclonal antibody is purchased from Dako company, Sigma company and company, Zhong Shan Golden Bridge respectively；Trizol reagent is purchased from Roche.
2. experimental method and result
2.1 oxetacaines (Oxethazaine) act on the induction replication initiation of hepatitis type B virus in HepAD38 cell
Research
1) Huh7 cell is pressed 5 × 104A/hole is inoculated in 24 porocyte culture plates, is cultivated in 37 DEG C of cell incubators
24h。
2) the plasmid pFlag-GAPDH of the expression Flag-GAPDH of Huh7 cell transient transfection CMV promoter control, and add
Medicine processing culture cell 3 days.
3) cell is collected, the expression feelings of the albumen with Flag label are detected by protein immunoblot (Western blot)
Condition, to detect active influence of the oxetacaine effect for CMV promoter.
As a result as shown in Figure 2.Fig. 2 is marked the results show that comparing oxetacaine with the control of not dosing group and having no effect on band flag
The expressing quantity of the GAPDH of label (expressing quantity of internal reference albumen β-actin is still kept constant).This also indicates that former times card difficult to understand
Because having no effect on the HBV virus replication starting under protein expression and its induction under CMV promoter control.
Influence of 2.2 oxetacaines (Oxethazaine) to HBV cccDNA
1) in HepAD38 cell, after tetracycline induces virus replication starting, tetracycline is removed, in nucleus gradually
Form the stable library cccDNA, the template continually replicated as HBV.
2) the HepAD38 cell after above-mentioned induction is pressed 4 × 104Cells/well is inoculated in 24 porocyte culture plates, and 37 DEG C
It is cultivated for 24 hours in cell incubator.
3) agent-feeding treatment cell: being diluted to various concentration for oxetacaine with DMEM/F12 culture medium, every group of 3 repetitions,
Various dose drug is added in tissue culture plate and cultivates cell, agent-feeding treatment 6 days.
4) cell is collected, extracts total HBV DNA and cccDNA (closed hoop DNA) intracellular respectively.Wherein, it directly takes out
Mentioning genomic DNA intracellular can be used to detection HBV total DNA；Extracting for cccDNA utilizes potassium chloride precipitating with high salt before this
Method removes most cell genomic dna, then (plasmid-safe ATP-dependent DNase is purchased with PSAD enzyme
From Epicentre company) (all DNA except selective degradation closed hoop double-stranded DNA) processing can remove a chain not
The HBV DNA replication dna intermediate products such as complete HBV gene group DNA (cyclic annular relaxed DNA, rcDNA), single-stranded HBV DNA, PSAD
Enzyme, which can substantially completely degrade, removes the ingredient of cccDNA in HBV total DNA, then can be obtained after purification more pure
cccDNA.The method analysis and research Antiviral Effect activity of obtained total HBV DNA and cccDNA sample quantitative PCR is simultaneously same
When evaluate agent-feeding treatment 6d cytotoxicity situation.
Real-time quantitative PCR the primer is as shown in NO:1~6 SEQ ID:
Primer (NCCC1): 5 '-CTCCCCGTCTGTGCCTTCT-3 ' (SEQ ID NO:5).
Primer (CCCAS2): 5 '-GCCCCAAAGCCACCCAAG-3 ' (SEQ ID NO:6).
Primer (HBV S144- forward primer): 5 '-ACTCACCAACCTCTTGTCCT-3 ' (SEQ ID NO:1).
Primer (HBV S144- reverse primer): 5 '-GACAAACGGGCAACATACCT-3 ' (SEQ ID NO:2).
Primer (GAPDH forward primer): 5 '-GAAGGTGAAGGTCGGAGTC-3 ' (SEQ ID NO:3).
Primer (GAPDH reverse primer): 5 '-GAAGATGGTGATGGGATTTC-3 ' (SEQ ID NO:4).
As a result as shown in Figure 3.Wherein, Fig. 3 A is shown plus oxetacaine handles 6 days cytotoxicities to HepAD38 cell
Situation；Fig. 3 B is shown: oxetacaine processing micro- rubs close to the 10 of maximal non-toxic concentration compared with non-agent-feeding treatment control group
You/liter when still the level of cccDNA is had not significant impact, but still have significant dose-dependant for HBV total DNA intracellular
Inhibitory effect (Fig. 3 C).Wherein, the 3TC in figure indicates that Lamivudine pharmaceutical group is added, and C is blank control group, i.e., non-dosing
Group.Fig. 3 is as the result is shown: oxetacaine can effectively lower the content of HBV total DNA intracellular, but not have to the content of cccDNA
Have a significant impact.The antiviral effect of oxetacaine is realized by the content for influencing the template cccDNA of hbv replication
's.
Research of 2.3 oxetacaines (Oxethazaine) to the effect of hepatitis type B virus RNA
1) HepAD38 cell is pressed 4 × 104Cells/well is inoculated in 24 porocyte culture plates, in 37 DEG C of cell incubators
Culture is for 24 hours.
2) agent-feeding treatment cell: being diluted to various concentration for oxetacaine with DMEM/F12 culture medium, every group of 3 repetitions,
Various dose drug is added in tissue culture plate, agent-feeding treatment cell 3 days.
3) cell extraction RNA is collected, with the effect of real-time quantitative PCR quantitative analysis drug RNAs various to HBV.
Real-time quantitative PCR the primer is as shown in NO:3~4 SEQ ID and NO:7~12 SEQ ID:
Primer (precoreRNA forward primer): 5'-TCTGCGCACCAGCACCATGCAAC-3'(SEQ ID NO:7).
Primer (precoreRNA reverse primer): 5'-GCGAAGGAAAGAAGTCAGAAGGC-3'(SEQ ID NO:8).
Primer (pgRNA forward primer): 5'-CTGGGTGGGTGTTAATTTGG-3'(SEQ ID NO:9).
Primer (pgRNA reverse primer): 5'-TAAGCTGGAGGAGTGCGAAT-3'(SEQ ID NO:10).
Primer (HBV total serum IgE forward primer): 5'-CCGTCTGTGCCTTCTCATCTGC-3'(SEQ ID NO:11).
Primer (HBV total serum IgE reverse primer): 5'-ACCAATTTATGCCTACAGCCTCC-3'(SEQ ID NO:12).
Primer (GAPDH RNA forward primer): 5'-GAAGGTGAAGGTCGGAGTC-3 ' (SEQ ID NO:3).
Primer (GAPDH RNA reverse primer): 5 '-GAAGATGGTGATGGGATTTC-3 ' (SEQ ID NO:4).
Experimental result is as shown in Figure 4.Wherein, the 3TC expression addition Lamivudine pharmaceutical group in figure, C blank control group, i.e.,
Non- dosing group.Fig. 4 is shown: compared with not dosing control group, the oxetacaine of various concentration for HBV precore mRNA,
The content of pgRNA and HBV total serum IgE has no significant effect.This result also indicates that the active spy that both gets along well of the Anti-HBV activity of oxetacaine
The opposite sex inhibits HBV transcription related, and the selective degradation HBV RNA that also gets along well is related.
In conclusion above as the result is shown: the activity and inhibition HBV viral secretory of the Anti-HBV activity of oxetacaine inhibit
CMV promoter transcription, inhibition cccDNA in HepAD38 cell are formed and are degraded, inhibit HBV transcription, selective degradation HBV
RNA and inhibition HBV DNA synthesis are uncorrelated.
The research for the effect that 2.4 oxetacaines (Oxethazaine) assemble hepatitis type B virus nucleocapsid:
The result of above embodiments has been proven that the antivirus action of oxetacaine is not to occur in virus replication week
The synthesis and degradation of the HBV cccDNA of phase, viral DNA synthesis, the processes such as transcription of viral RNA and viral secretory, inventor just will
The stage that oxetacaine plays a role narrows down to virus assembly during this.And then inventor has inquired into oxetacaine pair
The effect of HBV nucleocapsid assembling.Experimentation is as follows:
1) HepAD38 cell is pressed 2 × 105Cells/well is inoculated in 6 porocyte culture plates plates, 37 DEG C of cell incubators
Middle overnight incubation.
2) agent-feeding treatment cell 72 hours.
3) 300 microlitres of bland cell lysates are added, lysate are collected after lysis at room temperature 30min into EP pipe, and be centrifuged
Remove cell fragment.Lysate sample is divided into various composition with 1% agarose gel electrophoresis, is then turned with capillary transfer
It moves on on nylon leaching film, transferring film buffer uses 10 × SSC, detects the antibody of the HBV nucleocapsid Anti-HBV activity core on filter membrane
(Dako) it detects.The filter membrane detected alkaline denaturation handles the viral DNA in release nucleocapsid, with Southern blot method
Detect the level of nucleocapsid inner virus DNA.
In addition with the method detection HBV core protein of western blot, (core protein forms nucleocapsid shell
Albumen), antibody (Dako) detection of the antibody Anti-HBV activity core of detection.
As the result is shown as shown in Figure 5.Fig. 5 is shown: oxetacaine is to the HBV in the nucleocapsid assembling of HBV and nucleocapsid
DNA has significant dose-dependent inhibitory effect.
3 oxetacaine of embodiment (Oxethazaine) is antiviral to nucleoside analog class Drug Resistance HBV mutant strain
Effect
1. experimental material
1.1 cells, plasmid and drug
Huh7 is source of people liver cancer cells；
Plasmid REZ31-9-1 (59#) contains 1.1 times of wild type HBV gene group sequences；Adefovirdipivoxil (ADV) drug resistance is prominent
Become strain expression plasmid REZ36-10-1 (61#), 1.1 times of HBV gene group sequences containing N236T mutation；Lamivudine
The dual anti-pharmacological property mutant strain expression plasmid REZ7-8-4 (70#) of (3TC) and Entecavir (ETV), contains L180M+M204V+
1.1 times of HBV gene group sequences that S202G tri- is mutated.The above plasmid is that Wuhan institute of viruses Chen Xinwen researcher give.
Oxetacaine is purchased from Sigma company, and Lamivudine (3TC) is NIH present；Adefovirdipivoxil (ADV) and Entecavir
(ETV) it is purchased from Dalian U.S. logical sequence Technology Co., Ltd..
1.2 reagent
DMEM culture medium and fetal calf serum (FBS) are purchased from GIBCO company；Lipofectamine
Lipofectamine2000 is purchased from Invitrogen company；DIG High Prime DNA Labeling and Detection
Starter Kit II is purchased from Roche company.
2. experimental method and result
In the present embodiment, inventor is mutated oxetacaine (Oxethazaine) to nucleoside analogue drugs drug resistance HBV
The effect of the duplication situation of strain is studied, and experimental procedure is as follows:
1) Huh7 cell is pressed 2 × 105A/hole is inoculated in 6 porocyte culture plates, is cultivated in 37 DEG C of cell incubators
24h。
2) expression plasmid that Huh7 cell has transiently transfected 1.1 times of HBV gene groups respectively (including wild type WT, contains
The adefovirdipivoxil drug-resistant mutation strain of N236T mutation is replaced containing the Lamivudine being mutated of L180M+M204V+S202G tri- and grace
Block Wei Shuan drug resistance mutant strain), it is separately added into after transfection oxetacaine (Oxethazaine), Lamivudine (3TC), A De
Fu Wei (ADV) and Entecavir (ETV) processing, agent-feeding treatment 3 days.
3) cell is collected, the DNA in intracellular HBV nucleocapsid is extracted, with the digoxigenin labeled of HBV DNA specificity
Probe, with the level of DNA in the method detection HBV nucleocapsid of Southern blot hybridization (Southern blot).
As a result as shown in Figure 6.Wherein, C blank control group, i.e., non-dosing group.Fig. 6 is shown: oxetacaine can significantly press down
The HBV DNA replication dna of wild type processed and two kinds of drug-resistant mutation strains.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (8)
1. the purposes of oxetacaine or its pharmaceutically acceptable salt in medicine preparation, the drug is for inhibiting hepatitis B
Virion assembling.
2. purposes according to claim 1, which is characterized in that the drug therapy or prevention hepatitis type B virus sense
Dye.
3. purposes according to claim 2, which is characterized in that the drug is for treating or preventing human hepatitis B
Virus infection.
4. a kind of for treating or preventing hepatitis b virus infected pharmaceutical composition characterized by comprising
Oxetacaine or its pharmaceutically acceptable salt are as active constituent；And
Second medicament, the second medicament are different from oxetacaine or its pharmaceutically acceptable salt and for treating or in advance
Prevent hepatitis b virus infected.
5. pharmaceutical composition according to claim 4, which is characterized in that further comprise pharmaceutically acceptable carrier.
6. pharmaceutical composition according to claim 4, which is characterized in that the second medicament include selected from it is following at least
It is a kind of: interferon, Peg-IFN alpha-2b, Lamivudine, adefovirdipivoxil, Entecavir, Sebivo, tenofovir.
7. pharmaceutical composition according to claim 4, which is characterized in that described pharmaceutical composition is in tablet, injection, powder
Agent, elixir, capsule, suspension, syrup, pill, sustained release preparation, controlled release preparation or nanometer formulation.
8. pharmaceutical composition according to claim 4, which is characterized in that described pharmaceutical composition is for treating or preventing
Human hepatitis B virus infection.
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| Method Development and Validation of Sucralfate and Oxetacaine in Bulk Drug and Formulation by Rp-Hplc;Sujitha Parimala S;《International Journal of Innovative Pharmaceutical Research》;20131231;第4卷(第4期);第349-356页 * |
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