CN106074488B - Benzene methoxycarbonyl group-Pidolidone is in the purposes for preparing BLyS antagonists - Google Patents
Benzene methoxycarbonyl group-Pidolidone is in the purposes for preparing BLyS antagonists Download PDFInfo
- Publication number
- CN106074488B CN106074488B CN201610408919.3A CN201610408919A CN106074488B CN 106074488 B CN106074488 B CN 106074488B CN 201610408919 A CN201610408919 A CN 201610408919A CN 106074488 B CN106074488 B CN 106074488B
- Authority
- CN
- China
- Prior art keywords
- blys
- methoxycarbonyl group
- glutamic acid
- benzene methoxycarbonyl
- taci
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- 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
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/27—Esters, e.g. nitroglycerine, selenocyanates of carbamic or thiocarbamic acids, meprobamate, carbachol, neostigmine
Landscapes
- Health & Medical Sciences (AREA)
- Emergency Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention discloses benzene methoxycarbonyl group L glutamic acid in the purposes for preparing BLyS antagonists, competitive ELISA the experimental results showed that：Benzene methoxycarbonyl group L glutamic acid can inhibit the combination of BLyS and TACI Fc.Inhibiting effect and the concentration of benzene methoxycarbonyl group L glutamic acid are proportionate.Benzene methoxycarbonyl group L glutamic acid can inhibit the combination of BLyS and BCMA Fc；Inhibiting effect and the concentration of benzene methoxycarbonyl group L glutamic acid are proportionate.Benzene methoxycarbonyl group L glutamic acid can be used as potential BLyS small molecular antagonists.
Description
Technical field
The invention belongs to biomedicine fields, are related to benzene methoxycarbonyl group-purposes of the Pidolidone as BLyS antagonists.
Background technology
Bone-marrow-derived lymphocyte stimulating factor (B lymphocyte stimulator, BLyS), also known as B cell activity factor (B
Cell activating factor belonging to the TNF family, BAFF), it is a member in TNF families.By
Monocyte and macrophage are persistently synthesized, are secreted.BLyS can be in conjunction with three kinds of receptors on B cell surface, B cell maturation antigen
(B cell mature antigen, BCMA), cross-film activator and CAML conjugates (Transmembrane activator
And CAML-interactor, TACI) and BAFF receptor 3 (BR3).After receptor combination BAFF, withering for mature B cell is reduced
It dies.If knocking out BAFF, the mature B cell in Mice Body lacks completely.Since BLyS is sent out in bone-marrow-derived lymphocyte activation, proliferation
Important function is waved, and the humoral immunity that the antibody that bone-marrow-derived lymphocyte generates is mediated is in numerous autoimmune diseases having found
It is in Central Position in the middle.It is now recognized that the overexpressions of BLyS in vivo and such as systemic erythema of certain autoimmune diseases
Generation, the development of the courses of disease such as lupus (SLE), rheumatoid arthritis (RA) are closely related.Therefore, to block BLyS biology work(
It can be strategy, inquire into BLyS and inhibit the autoimmune diseases such as antagonist for treating systemic loupus erythematosus and rheumatoid arthritis
In the external progress like a raging fire of research of B cell tumor disease.Currently, the research for the inhibitor of BLyS is main
It concentrates on developing BLyS Decoy receptors (decoy receptor), anti-BLyS antibody and the antagonistic peptide for having and neutralizing BLyS effects
[1-3].In March, 2011, U.S. FDA approval was by Human Genome Sciences Inc. (Human Genome Sciences) and Ge Lanshi-
Anti- BLyS antibody BENLYSTA (Belimumab) treatment system of SmithKline (GlaxoSmithKline) company cooperative research and development is red
Yabbi sore.This is over 50 years, and FDA ratifies to treat the drug of such disease for the first time.But protein inhibitor has as medicine
There is certain limitation, such as source is less and isolates and purifies that difficulty is big and medicine stability is poor, additionally there are oral
Bioavilability it is relatively low, it is high to the degradability of polypeptide drug to be primarily due to internal various enzymes, to lead to this one kind
The effect half-life period of drug greatly shortens.Therefore, exploitation micromolecular compound has important as the research of BLyS inhibitor
Realistic meaning.Currently, there has been no benzene methoxycarbonyl group-Pidolidone the purposes for preparing BLyS antagonists report.
1.Jian Sun* (Sun Jian), Zhou Lin, Jiannan Feng, Yan Li, Beifen Shen. (2008)
BAFF-targeting therapy,a promising strategy for treating autoimmune
diseases.Eur J Pharmacol 597:1–5.
2.Yacong Zhao, Xiafei Hao, Jiannan Feng, Beifen Shen, Jing Wei* (Wei Jing),
Jian Sun* (Sun Jian) (2015), The comparison of BLyS-binding peptides from phage
display library and computer-aided design on BLyS–TACI interaction.Int
Immunopharmacol,24:219–223.
3.Xiafei Hao,Yanfeng Zhu,Chang Zheng,Xuegang Fu,Jiannan Feng,Beifen
Shen, Jing Wei* (Wei Jing), Jian Sun* (Sun Jian) .A comparison of biological activity of B
lymphocyte stimulator(BLyS)antagonist peptibodies and the elucidation of
possible BLyS binding sites.Protein&Peptide Letters,2016,23,17-23
4.Xuegang Fu, Liyan Xuan, Yuzhe Wang, Jing Wei* (Wei Jing), Jian Sun* (Sun Jian)
.Molecular mechanism of the affinity interactions between BAFF and its
peptides by molecular simulations.Protein&Peptide Letters,2015,22,992-999.
Invention content
It is short of money in preparation BLyS the purpose of the present invention is overcoming the deficiencies of the prior art and provide benzene methoxycarbonyl group-Pidolidone
The purposes of anti-agent.
Technical scheme of the present invention is summarized as follows：
Benzene methoxycarbonyl group-Pidolidone is in the purposes for preparing BLyS antagonists
Benzene methoxycarbonyl group-Pidolidone is hereinafter simply referred to as CBz-L- glutamic acid.
Advantages of the present invention：
1. having for BLyS combinations TACI and significantly inhibiting effect
Competitive ELISA the experimental results showed that：CBz-L- aminoglutaric acid concentrations can inhibit 1.26 ± 3.79% in 1mg/mL
The combination of BLyS and TACI-Fc；It can inhibit the combination of 59.35 ± 11.87%BLyS and TACI-Fc in 3mg/mL；
It can inhibit the combination of 74.13 ± 6.78%BLyS and TACI-Fc when 5mg/mL.The concentration of inhibiting effect and CBz-L- glutamic acid
It is proportionate.
The micromolecular compound (benzoic acid) of non-binding BAFF, i.e. negative control, do not have the interaction of BLyS and TACI
There is obvious inhibiting effect and there is no dose-dependence.Prove that the inhibiting effect of CBz-L- glutamic acid is special and effective.
2. having for BLyS combinations BCMA and significantly inhibiting effect
Competitive ELISA the experimental results showed that：CBz-L- aminoglutaric acid concentrations can inhibit 5.63 ± 0.88% in 1mg/mL
The combination of BLyS and BCMA-Fc；It can inhibit the combination of 56.70 ± 7.90%BLyS and BCMA-Fc in 3mg/mL；In 5mg/
It can inhibit the combination of 82.80 ± 3.98%BLyS and BCMA-Fc when mL.Inhibiting effect and the concentration of CBz-L- glutamic acid are in just
It is related.
Negative control compound (benzoic acid) is to the no obvious inhibiting effect of the interaction of BLyS and BCMA and does not have agent
Measure dependence.Prove that the inhibiting effect of CBz-L- glutamic acid is special and effective.
Description of the drawings
Fig. 1 is the Key residues of BLyS bind receptors ,+indicate Key residues.
Fig. 2 is the molecular surface and Key residues of BLyS binding pockets, and D1 indicates that conservative calmodulin binding domain CaM, D2 indicate special
Property calmodulin binding domain CaM.
Fig. 3 is virtual screening flow chart.
Fig. 4 is CBz-L- glutamic acid (benzene methoxycarbonyl group-Pidolidone) structural formula.
Fig. 5 is the pattern of CBz-L- glutamic acid and the interaction of BLyS.
Fig. 6 is the combination that CBz-L- glutamic acid inhibits TACI to BLyS.* represents p<0.01；* 0.01 is represented<p<
0.05。
Fig. 7 is the combination that CBz-L- glutamic acid inhibits BCMA to BLyS.* represents p<0.01.
Specific implementation mode
With reference to specific embodiment, the present invention is further illustrated.It is numerous research elaborated BLyS with by
The possibility pattern and key amino acid of body interaction.We, by molecular dynamics simulation, are based on BLyS in previous work
It is probed by the Interactions Mode of computer simulation BLyS and three of them natural receptor with the crystal structure of its receptor BCMA
It participates in the Key residues for guarding sexual reaction and affinity selectivity.On this basis, " key-lock-principle " is utilized, for pass
Key calmodulin binding domain CaM carries out commercialization chemical small molecule database and carries out molecule virtual screening, and prediction obtains a series of with potential suppression
The micromolecular compound of making obtains its entity and carries out active determination in vitro discovery, binary acid compounds dibenzoyl
Tartaric acid has the function of certain inhibition BLyS combinations TACI and BCMA.There is the new drug gateway drug of potential applicability in clinical practice for exploitation
Object has established experimental study basis.
With reference to specific embodiment, the present invention is further illustrated.The embodiment of the present invention is to make this field
Technical staff can implement, but the present invention is not imposed any restrictions.Disclosing for common agents therein is to make this
The technical staff in field preferably implements the present invention, but is not imposed any restrictions to the present invention.
BLyS albumen：Sun Jian, Li Yan, Feng Jiannan, Sun Yingxun, Hu Meiru, Shen are put forth energy again, the stimulation of human soluble bone-marrow-derived lymphocytes
The clone of factor gene and expression [J] the cells in Escherichia coli and molecular immunology magazine, 2006, (2) SEQ ID
Shown in NO.8.
TACI-Fc albumen：Ji Lijun, white Wu Ren scheme refined, Chai Lin, Sun Jian .TACI-Fc fusion proteins it is gene constructed, former
Nuclear expression and Biological Activity Identification [J] biotechnologys, 2013, shown in (3) .SEQ ID NO.9
BCMA-Fc albumen：Sun Jian, Feng Jiannan, Linzhou, Li Yan, Shen put forth energy again CAD solubility BLyS by
Body, eBCMA-Fc fusion proteins and its expression Chinese biologicals chemistry in Escherichia coli and molecular biosciences journal, 2008,24
(2) shown in SEQ ID NO.9.
Fc albumen：Research [D] University Of Tianjin of the Wu Zhen .BCMA-Fc as potential drug, 2012SEQ ID NO.10 institutes
Show.
Coating buffer：33.6g sodium bicarbonates and 63.6g natrium carbonicum calcinatums are weighed, is put it into beaker, is inwardly added
600ml distilled water, stirring to solute are all dissolved, and add in distilled water constant volume to 1L.It places it at 4 DEG C and preserves.
The preparation of PBST：It takes 250 μ l Tween-20s to be added in 1 × PBS of 500ml, is uniformly mixed to complete fusion.In room
Temperature is lower to be preserved.
The preparation of 5% skimmed milk power：Skimmed milk power 1g is weighed, is dissolved under stirring in 10ml 0.01M PBS, adds PBS fixed
Hold to 20ml, in 4 DEG C of preservations
TMB developing solutions：Take 4.95ml substrate buffer solutions, 0.05ml 1%TMB liquid storages that 5 μ l, 30% peroxides are added before use
Change hydrogen.
1%TMB liquid storages：1g TMB are weighed, is completely dissolved under stirring in 80ml DMSO, DMSO is added to be settled to 100ml,
It is stored in 4 DEG C.
Substrate buffer solution (pH 5.0)：24.3ml 0.1M citric acids, 25.7ml 0.2M disodium hydrogen phosphates is taken to be added about
40ml water adjusts pH to 5.0, adds water and be settled to 100ml, be stored in room temperature.TMB working solutions (now with the current).
Embodiment 1：With computer virtual sifting technology, BLyS correlation micromolecular compounds are filtered out.
Protein structural information obtains：From Protein Date Bank Protein Data Banks (http://www.rcsb.org/
Pdb BLyS protein structures (PDB ID are downloaded in)：1OQD and 1OQE).Using computational methods such as molecular dynamicses, from molecular level
On probe into the interaction mechanism of BLyS and its natural receptor (BCMA, TACI, BR3).According to each amino acid residue to combining
Energy contribution, determines its Key residues.Fig. 1 and Fig. 2 respectively illustrates the key effect residue of BLyS target spot natural receptors, with
And the binding pocket of BLyS target spots and the relevant information of Key residues.
The acquisition of small molecule database：Small molecule database contains nearly ten thousand different compounds of structure, main source
In two approach：Small molecule in natural products database or commercial small molecule database, the seriation that project is combined into
Close object.Commercial small molecule database refers mainly to lark prestige (J＆K), AlfaAesar (Alfa-Aesar), Sigma-Aldrich
(Sigma-Aldrich) three Reagent Company websites obtain.Three-dimensional small molecule structure is carried out after adding hydrogen, power-up lotus processing, it will
File is preserved into pdbqt file formats, carries out further molecular Docking Study.
Virtual screening：By 4.2 softwares of AutoDock, by all small molecules in database and the activated centres BLyS region
Screening docking is carried out to calculate.During molecular docking, three-dimensional small molecule structure data are placed at the active site of BLyS one by one,
By continuing to optimize the position of small molecule model, the dihedral angle of conformation, the rotatable key of intramolecule, finds compound and make with BLyS
Best conformation, and predict its binding pattern.Analysis docking according to the small molecule prediction provided as a result, combine score value, investigation
The matching degree of compound and BLyS combinations center, and with key amino acid correlation situation, filter out it is potential have BLyS
The small molecule candidate compound of affinity.Score value is bigger, and the affinity of representation compound and BLyS are higher, i.e., compound may have
Preferable affinity.The compound for selecting overall merit best obtains chemical entities, and carries out ELISA experiments.It is retouched in Fig. 3
The flow chart of virtual screening is stated.
Embodiment 2：Computer analyzes small molecular antagonists and BLyS binding patterns.
Using computer molecular docking method, CBz-L- glutamic acid has been inquired into conjunction with Chimera softwares (structure is referring to Fig. 4)
With BLyS Study on Molecular Mechanism, itself and the BLyS critical amino acid residues being mutually distinguishable and key interactions (Fig. 5) are analyzed.
It was found that a phenyl ring of CBz-L- glutamic acid is inserted into the conservative hydrophobic pocket of BLyS, formed with His69, Leu70 and Ile92
Hydrophobic interaction, and form π-cation with Arg124 and Arg90 residue side chains and act on, it is common reinforce CBz-L- glutamic acid with
The combination of BLyS；Carboxyl on two side chains forms salt bridge with Arg90；In oxygen and amino acid carboxyl on benzyloxycarbonyl group
Ketonic oxygen and the Key residues Arg124 of BLyS form interaction of hydrogen bond, the OH and Y65 in amino acid carboxyl forms hydrogen bond.
Embodiment 3：Inhibiting effect of the competitive ELISA analysis of compounds for BLyS combination TACI/BCMA albumen.
If compound CBz-L- glutamic acid can be with TACI/BCMA competitive binding BLyS active pockets, the change being added
BLyS combinations TACI/BCMA can be inhibited by closing object CBz-L- glutamic acid；On the contrary, if compound CBz-L- glutamic acid does not combine
BLyS, also just there is no competed with TACI/BCMA.Compound CBz-L- glutamic acid is then added not have BLyS combinations TACI/BCMA
Have an impact.Therefore, it is tested by Competitive assays ELISA, BLyS and receptor TACI is blocked to test small molecule CBz-L- glutamic acid
The effect combined with BCMA.
First, routinely ELISA method is the ELISA that BLyS is combined with TACI-Fc/BCMA-Fc.Determine Competitive assays
The concentration of TACI-Fc/BCMA-Fc is used in ELISA experiments.It is final to determine that concentration is 10 μ g/mL.The tool of Competitive assays ELISA
Steps are as follows for gymnastics work：
A) it is coated with：The BLyS of 20g/mL presses 1 with coating buffer:1 dilution, prepares the BLyS liquid of 10 μ g/mL.It will be diluted
BLyS liquid is coated with 96 orifice plates by 50 microlitres/hole.4 DEG C overnight.
B) it after board-washing, is closed 2 hours with 5% skimmed milk power room temperature.
C) board-washing, then the CBz-L- glutamic acid aqueous solutions of the TACI-Fc of 10g/mL or BCMA-Fc and different volumes are mixed
It closes, prepares the mixed solution that micromolecular compound CBz-L- glutamic acid final concentration gradients are 0,1,3 and 5mg/mL, 50 are added per hole
Microlitre.It 37 DEG C, incubates 1 hour.Not plus the mixed solution of small molecule solution is as blank control group.Benzoic acid is negative control
Close object.
D) HRP label goat-antis people 2 anti-(by specification dilution) is added in board-washing.
E) TMB chromogenic reagents are added after board-washing.
F) reaction is terminated, 450 nanometers of OD values are surveyed.Compound competes the interaction of BLyS and TACI-Fc/BCMS-Fc
Inhibition calculated with following formula：Inhibiting rate %=[(blank control group OD450- experimental group OD450)/blank control groups
OD450] * 100% (inhibiting rate is shown in Fig. 6 and Fig. 7).
Claims (1)
1. benzene methoxycarbonyl group-Pidolidone is in the purposes for preparing bone-marrow-derived lymphocyte stimulating factor (BLyS) antagonist.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610408919.3A CN106074488B (en) | 2016-06-08 | 2016-06-08 | Benzene methoxycarbonyl group-Pidolidone is in the purposes for preparing BLyS antagonists |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610408919.3A CN106074488B (en) | 2016-06-08 | 2016-06-08 | Benzene methoxycarbonyl group-Pidolidone is in the purposes for preparing BLyS antagonists |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106074488A CN106074488A (en) | 2016-11-09 |
| CN106074488B true CN106074488B (en) | 2018-07-27 |
Family
ID=57229143
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610408919.3A Expired - Fee Related CN106074488B (en) | 2016-06-08 | 2016-06-08 | Benzene methoxycarbonyl group-Pidolidone is in the purposes for preparing BLyS antagonists |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106074488B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004113363A2 (en) * | 2003-06-19 | 2004-12-29 | The Nottingham Trent University | Dipeptide transglutaminase inhibitors and methods of using the same |
| CN101969920A (en) * | 2008-03-12 | 2011-02-09 | 株式会社资生堂 | Parakeratosis inhibitor, pore shrinking agent or agent for preventing or improving rough skin, and composition for external use on the skin containing the same |
-
2016
- 2016-06-08 CN CN201610408919.3A patent/CN106074488B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004113363A2 (en) * | 2003-06-19 | 2004-12-29 | The Nottingham Trent University | Dipeptide transglutaminase inhibitors and methods of using the same |
| CN101969920A (en) * | 2008-03-12 | 2011-02-09 | 株式会社资生堂 | Parakeratosis inhibitor, pore shrinking agent or agent for preventing or improving rough skin, and composition for external use on the skin containing the same |
Non-Patent Citations (2)
| Title |
|---|
| BAFF-targeting therapy, a promising strategy for treating autoimmune diseases;Jian Sun et al.;《European Journal of Pharmacology》;20080830;第597卷;1-5 * |
| The B cell: a new therapeutic target in rheumatoid arthritis and other autoimmune diseases;Xavier Mariette;《Joint Bone Spine》;20040301;第71卷;357-360 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106074488A (en) | 2016-11-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lu et al. | Harnessing allostery: a novel approach to drug discovery | |
| Luck et al. | Structure and dynamics of drug carriers and their interaction with cellular receptors: focus on serum transferrin | |
| Pulido et al. | Design of a true bivalent ligand with picomolar binding affinity for a G protein-coupled receptor homodimer | |
| Almerico et al. | A3 adenosine receptor: Homology modeling and 3D-QSAR studies | |
| Fulton et al. | Conformationally constrained peptides target the allosteric kinase dimer interface and inhibit EGFR activation | |
| CN102811729B (en) | Materials and methods related to sodium/potassium adenosine triphosphase and SRC | |
| Baghban et al. | Design and in silico evaluation of a novel cyclic disulfide-rich anti-VEGF peptide as a potential antiangiogenic drug | |
| Jin et al. | Moving toward understanding eukaryotic chemotaxis | |
| Muratspahić et al. | Design and structural validation of peptide–drug conjugate ligands of the kappa-opioid receptor | |
| CN106074488B (en) | Benzene methoxycarbonyl group-Pidolidone is in the purposes for preparing BLyS antagonists | |
| Harikumar et al. | Molecular basis for benzodiazepine agonist action at the type 1 cholecystokinin receptor | |
| Desai et al. | Molecular mechanism of action of triazolobenzodiazepinone agonists of the type 1 cholecystokinin receptor. Possible cooperativity across the receptor homodimeric complex | |
| CN105919989B (en) | (2R, 3R)-dibenzoyl tartaric acid is in the purposes for preparing BLyS antagonist | |
| CN101157951B (en) | Method for assessing proliferation inhibiting effect of inhibitor, and method for determining sensitivity of tumor cell to inhibitor | |
| Kubota et al. | Chemogenetic approach using Ni (II) complex–agonist conjugates allows selective activation of class A G-protein-coupled receptors | |
| CN102341708B (en) | Screening method for epidermal growth factor receptor tyrosine kinase inhibitor and inhibitors identified using same | |
| CN106074476A (en) | Palmoxiric acid is preparing the purposes of BLyS antagonist | |
| Zhang et al. | Discovery of novel antagonists of human neurotensin receptor 1 on the basis of ligand and protein structure | |
| Chen et al. | Integrated in silico–in vitro rational design of oncogenic EGFR-derived specific monoclonal antibody-binding peptide mimotopes | |
| Matsunaga et al. | Synergistic Ca2+ and Cu2+ requirements of the FGF1–S100A13 interaction measured by quartz crystal microbalance: An initial step in amlexanox-reversible non-classical release of FGF1 | |
| Selvanesan et al. | Lymphocyte antigen 6K signaling to aurora kinase promotes advancement of the cell cycle and the growth of cancer cells, which is inhibited by LY6K-NSC243928 interaction | |
| Shapira et al. | Discovery of novel spike/ACE2 inhibitory macrocycles using in silico reinforcement learning | |
| CN104193806B (en) | BLyS antagonistic peptide SS12, fusion protein SS12-Fc containing antagonistic peptide and gene | |
| Guidetti et al. | Molecular Determinants of EphA2 and EphB2 Antagonism Enable the Design of Ligands with Improved Selectivity | |
| Sochacka et al. | Characterization of 6-Mercaptopurine binding site on human α1-acid glycoprotein (orosomucoid) using molecular docking |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180727 Termination date: 20210608 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |