CN104257655B - Compound MEAN purposes in the medicine that preparation suppresses HCV to replicate - Google Patents
Compound MEAN purposes in the medicine that preparation suppresses HCV to replicate Download PDFInfo
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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
- A61K31/47—Quinolines; Isoquinolines
- A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
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Abstract
The present invention relates to pharmaceutical technology field, be specially compound MEAN purposes in the medicine that preparation suppresses HCV to replicate。The purposes in the medicine that preparation suppresses HCV to replicate of the compound MEAN shown in formula (1)。Described medicine is the suppressor gene type 2 HCV medicine replicated。And compound MEAN combines IFN-α/RBV/ITX 5061 and works in coordination with the duplication of suppressor gene 2 type HCV。MEAN can the duplication of suppressor gene 2 type hepatitis C (HCV)。
Description
Technical field
The present invention relates to pharmaceutical technology field, be specially compound MEAN purposes in the medicine that preparation suppresses HCV to replicate。
Background technology
Hepatitis C is to be infected, by hepatitis C virus (HepatitisCvirus, HCV), the global infectious disease caused, is also the one of the main reasons causing liver cirrhosis and hepatocarcinoma。About the HCV infection person of 80% can develop into chronic hepatitis。At present, Peg-IFN alpha-2b (PEGIFN) combines the standard scheme that ribavirin (RBV) is treatment chronic hepatitis C (CHC), but only have about 50% patient and can obtain continued viral response (SVR), and there is problems such as using taboo, individual variation and drug side effect。It would therefore be highly desirable to develop new safely and effectively alternative medicine。
Micromolecular compound is suppressed the HCV development replicating research to play great function by HCV replicon in-vitro culture model and being successfully established of infection model。At present, the clone's system being usually used in building HCV in-vitro culture model is JFH1 strain。Conventional cultivation cell is human hepatoma cell strain Huh-7 and derived cell system Huh-7.5 and Huh-7.5.1 thereof。The chimeric strain pFL-JC1 of HCV2aJFH1 and the J6 built subsequently confirms that transfecting Huh-7.5 cell than JFH1 strain has higher duplicating efficiency。
Compound MEAN (6-methoxyethylamino-numonafide) is a newly synthesized class numonafide compound, the synthesis path document of this compound has been reported (JohnT.Norton, Anti-CancerDrugs2008,19 (11)), but its antiviral activity has no report, the present invention provides the new application of compound MEAN (6-methoxyethylamino-numonafide) anti-HCV activity。
The structural formula of compound MEAN (6-methoxyethylamino-numonafide) is as follows:
Molecular weight: 342.18。
Summary of the invention
It is an object of the invention to open MEAN application in therapeutic gene 2 type hepatitis C (HCV) medicine。
The purposes in the medicine that preparation suppresses HCV to replicate of the compound MEAN shown in formula (1)。
Described medicine is the medicine of suppressor gene 2 type hepatitis C virus replication, and its preferred dose is 5 μMs。
Compound MEAN suppresses the medicine of the duplication of HCVE2 mutant JC1/N415D or JC1/G451R in preparation
Purposes in thing。
Compound MEAN and the IFN-α purposes in the medicine that preparation suppresses HCV to replicate。In described medicine, MEAN and IFN-α usage ratio are 1:2。
Compound MEAN and RBV purposes in the medicine that preparation suppresses HCV to replicate。
Compound MEAN and ITX5061 purposes in the medicine that preparation suppresses HCV to replicate。In described medicine, MEAN and ITX5061 usage ratio is 1:8。
Described medicine is the suppressor gene 2 type HCV medicine replicated。
Accompanying drawing explanation
Fig. 1 MEAN cytotoxicity analysis, wherein after 22 hours curve downward be 20 μMs。
Fig. 2 MEAN HCV-Ab IgG EC50 analyzes
Fig. 3 MEAN HCV-Ab IgG effect analysis
Fig. 4 MEAN inhibitory action to HCVRNA and HCVCoreprotein
The effect of anti-E2MutantN415D and G451R of Fig. 5 MEAN
Fig. 6 MEAN works in coordination with the effect of the duplication of suppressor gene 2 type HCV respectively with IFN-α/RBV/ITX5061
Detailed description of the invention
By concrete drawings and Examples, the present invention will be described in detail in order to make it easy to understand, following。It needs to be noted, instantiation and accompanying drawing are merely to explanation, the present invention according to illustrating herein, can be made various correction and change by obvious those of ordinary skill in the art within the scope of the invention, and these are revised and change and also include in the scope of the present invention。
In the following example, method therefor is if no special instructions, is conventional method。Material required in following example or reagent, be market if no special instructions and buy。
The synthesis path document of compound MEAN (6-methoxyethylamino-numonafide) has been reported (JohnT.Norton, Anti-CancerDrugs2008,19 (11))。
Embodiment 1 drug cytotoxicity analysis
Cell is cultivated
Huh-7.5.1 cell with the DMEM culture medium containing 10% hyclone 37 DEG C, the incubator of 5%CO2 saturated humidity is cultivated。Culture medium is added the penicillin of 100U/ml and the streptomycin of 100ug/ml。
The preparation of HCVRNA transcription
Taking 15 μ gpFL-JC1 plasmids, carry out endonuclease reaction with restricted enzyme Xba I, hatch 2 hours for 37 DEG C, agarose gel electrophoresis detects whether total Linearization afterwards。Digestion products mung-bean nuclease and protease K digesting are processed, then with phenol chloroform method extracting and purifying DNA, then through agarose gel electrophoresis detection also quantitatively。Then take 2 μ gDNA templates, operate to specifications with T7 in vitro transcription test kit and carry out in vitro transcription。The rna transcription body obtained detects through agarose gel electrophoresis and measures concentration。
The foundation of HCV cell infection model
Transfect first 1 day, with 1 × 107Individual/hole density inoculation 10cm culture dish, cell fusion degree is 70%～80%。During transfection, take 10 μ gHCVRNA transcriptions, import to the hepatoma cell line Huh7.5.1 supporting that HCV replicates with the method for 260V and 25 milliseconds of pulse length folk prescription ripple electrotransfections。Collecting cell conditioned medium, the centrifugal 5min of 1500g is used for infecting after removing cell debris。
Cytotoxicity experiment
With 5 × 103Huh7.5.1 is inoculated in 96 porocyte chip boards by individual/hole density, 0.313 μM, 0.625 μM, 1.25 μMs, 2.5 μMs, 5 μMs, and the MEAN effect Huh7.5.1 cell 72h of 10 μMs and 20 μMs, real-time n cell analytical technology method detects its cytotoxicity。
As it is shown in figure 1, MEAN does not affect 10 μMs of (containing 10 μMs) following on cell proliferation。
Embodiment 2MEAN HCV-Ab IgG pharmacodynamic analysis
External HCV-Ab IgG effect analysis
After Huh7.5.1 cell is inoculated in 96 orifice plate overnight incubation with 5000 every holes, wild-type virus JC1-luc is to give 0.625 μM after MOI0.01 infection cell 24h, 1.25 μMs, 2.5 μMs, the MEAN of 5 μMs, adopts microplate luminometer (TurnerBiosystems) detection by quantitative uciferase activity after 48 hours。And by monitoring the inhibitory activity of the expression checking MEAN of HCVRNA and albumen in the Huh7.5.1 cell infecting virus。
Quantitative fluorescent PCR
Utilize HCVRNA relative quantity in fluorescence quantitative PCR detection cell。Collecting cell after trypsinization, often pipe addition Trizol extracts total serum IgE according to operating instruction, and ultraviolet spectrophotometer measures concentration。Taking 4 μ lRNA and first remove genomic DNA according to Reverse Transcriptase kit operating instruction, then carry out reverse transcription reaction, reaction system is 20 μ l。The cDNA of synthesis is as real-time quantitative PCR template, and reaction system includes each 0.4 μ l of positive antisense primer, the RoxII0.4 μ l of 2 × SYBRGreenTaq reactant liquor 10 μ l, HCV or GAPDH, sterile purified water 6.8 μ l and cDNA template 22 μ l。PCR reaction condition is 95 DEG C of 30s；95 DEG C of 5s, 60 DEG C of 30s, totally 40 circulations。Primer for detecting HCV is positioned at HCV5 ' UTR, upstream sequence: 5 '-GCGTTAGTATGAGTGTCGTG-3 ', downstream sequence: 5 '-TCGCAAGCACCCTATCAG-3 '；Reference gene glyceraldehyde 3-phosphate dehydro-genase (GAPDH) amplimer, upstream: 5 '-GAAGGTGAAGGTCGGAGTC-3 ', downstream: 5 '-GAAGATGGTGATGGGATTTC-3 '。All samples genes of interest relative expression levels calculates after internal standard gene GAPDH homogenization processes。
WesternBlot detects
WesternBlot detects D8L expression。Extract total protein of cell and carry out quantitative analysis according to the operating instruction of BCA protein quantification test kit。Taking above-mentioned total protein of cell 40 μ g uses SDS-PAGE to separate and transfer on nitrocellulose filter。After 5% defatted milk powder is closed, by film and antibody HCVCore (Abcam) and 4 DEG C of overnight incubation of β-actin (Cellsignaling), two anti-hatch 1 hour after with chemical illuminating reagent (Pierce) detection。
Fig. 2 shows, the EC50 that MEAN suppressor gene type 2HCV replicates is 2.36 ± 0.29 μMs；IFN-α is 4.83 ± 0.57U/ml；RBV does not individually have antiviral effect。
Fig. 3 shows, MEAN replicates in dose-dependently suppressor gene 2 type HCV, it is suppressed that effect is with 5 μMs of the bests。
Fig. 4 shows, MEAN suppresses the expression of HCVRNA and Core albumen。
Result above shows, MEAN can suppress the duplication of suppressor gene 2 type HCV, it is preferable that dosage is 5 μMs。
The anti-E2 mutant JC1/N415D of embodiment 3MEAN or JC1/G451R effect
In Vitro Anti E2 mutant JC1/N415D or JC1/G451R effect analysis
After Huh7.5.1 cell is inoculated in 96 orifice plate overnight incubation with 5000 every holes, mutant virus JC1/N415D or JC1/G451R, to give the MEAN of Concentraton gradient after MOI0.01 infection cell 24h, adopts microplate luminometer (TurnerBiosystems) detection by quantitative uciferase activity after 48 hours。
As it is shown in figure 5, MEAN also can mutation inhibiting strain JC1/N415D and JC1/G451R virus duplication。
Embodiment 4MEAN combines IFN-α/RBV/ITX5061 In Vitro Anti JC1 effect
External associating pharmacodynamic analysis
After Huh7.5.1 cell is inoculated in 96 orifice plate overnight incubation with 5000 every holes, wild-type virus JC1-luc give after MOI0.01 infection cell 24h Concentraton gradient MEAN combine IFN-α/RBV/ITX5061 (MEAN combine IFN-α usage ratio for 1:2；When MEAN combines RBV, individually there is no antiviral effect because of RBV, therefore RBV consumption is fixed as 17 μMs；It is 1:8 that MEAN combines ITX5061 usage ratio), adopt microplate luminometer (TurnerBiosystems) detection by quantitative uciferase activity after 48 hours。
As shown in Figure 6, MEAN and IFN-α/RBV/ITX5061 can work in coordination with the duplication of suppressor gene 2 type HCV。
Showing based on the above results, MEAN can the duplication of suppressor gene type 2 hepatitis C (HCV)。
Embodiment 5
Material and method: in 96 porocyte chip boards, the MEAN effect Huh7.5.1 cell 72h of Concentraton gradient (DMSO, 0.25 μM, 0.5 μM, 1 μM, 2 μMs, 4 μMs, 8 μMs, 16 μMs), the cytotoxicity of real-time n cell analytical technology detection MEAN。Transfection Huh7.5.1 cell after HCVJC1 plasmid external preparation HCVRNA transcription, collect cell conditioned medium and again hatch Huh-7.5.1 cell to set up HCV infection cell model (Huh7.5.1-JC1), after infection, 24h gives Concentraton gradient (0.625,1.25,2.5,5,10 μMs) MEAN process after cell 24h and be changed to fresh complete medium, detect luciferase level after 48h and judge MEAN HCV-Ab IgG effect。And the HCV-Ab IgG effect of the level verification MEAN of HCVRNA and core protein is detected respectively by Q-PCR and westernblot。Give after HCVIRES luciferase reporter gene plasmid transfection Huh7.5.1 cell 24h to change liquid after MEAN processes 24h, after 48h, detect the Dual-Luciferase level research MEAN impact on the translation that HCVIRES mediates。Additionally, process Huh7.5.1-JC1 with MEAN associating IFN α or ribavirin to explore its joint effect。The shadow that PTB expression and karyon-kytoplasm thereof are shuttled back and forth by Westernblot, immunofluorescence experiment and heterokaryon experiment detection MEAN。
Result: within RTCA result shows MEAN10 μM, (including 10 μMs) on cell proliferation does not affect (p > 0.05)。Luciferase reporter gene experiment confirms that MEAN can efficiently suppress HCV to replicate (p < 0.05), and medium effective concentration (EC50) is 2.5 μMs。The suppression HCVRNA of Q-PCR and westernblot result display MEAN dose dependent and core protein level, further demonstrate that MEAN is to the HCV depression effect replicated。Luciferase reporter gene experiment confirms the MEAN Translational repression DeGrain (p > 0.05) that HCVIRES is mediated。Medication combined experiment finds that MEAN has cooperative effect with interferon-ALPHA (IFN α) and ribavirin。WesternBlot, immunofluorescence experiment and heterokaryon experiment confirm that MEAN can suppress PTB karyon-kytoplasm transposition, but it can not be suppressed to express。
Conclusion: MEAN suppresses the HCV effect replicated by suppressing the karyon-kytoplasm transposition of PTB to play。
Claims (7)
1. the purposes in preparing the suppressor gene 2 type HCV medicine replicated of the compound MEAN shown in formula (1)
2. purposes according to claim 1, it is characterised in that: described medicine is the medicine of the duplication suppressing HCVE2 mutant JC1/N415D or JC1/G451R。
3. compound MEAN and the IFN-α purposes in preparing the suppressor gene 2 type HCV medicine replicated。
4. purposes according to claim 3, it is characterised in that: in described medicine, MEAN and IFN-α usage ratio are 1:2。
5. compound MEAN and RBV purposes in preparing the suppressor gene 2 type HCV medicine replicated。
6. compound MEAN and ITX5061 purposes in preparing the suppressor gene 2 type HCV medicine replicated。
7. purposes according to claim 6, it is characterised in that: in described medicine, MEAN and ITX5061 usage ratio is 1:8。
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| CN201410439186.0A CN104257655B (en) | 2014-08-31 | 2014-08-31 | Compound MEAN purposes in the medicine that preparation suppresses HCV to replicate |
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