CN109528700A - Application of the small molecule compound QW07 in preparation treatment prostatic disorders drug - Google Patents
Application of the small molecule compound QW07 in preparation treatment prostatic disorders drug Download PDFInfo
- Publication number
- CN109528700A CN109528700A CN201711123382.7A CN201711123382A CN109528700A CN 109528700 A CN109528700 A CN 109528700A CN 201711123382 A CN201711123382 A CN 201711123382A CN 109528700 A CN109528700 A CN 109528700A
- Authority
- CN
- China
- Prior art keywords
- androgen receptor
- formula
- application
- cancer
- cell
- 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.)
- Pending
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
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
- A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
Landscapes
- Health & Medical Sciences (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)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses application of one kind small molecule compound QW07 as shown in formula (I) in preparation treatment prostate cancer disease drug, the compound can bind directly androgen receptor NTD binding domain, inhibit androgen receptor transcription complex formation and its and downstream gene promoter and enhancer combination, inhibit the transcription of overall length and truncated-type androgen receptor to downstream gene, the selective depression to androgen receptor positive cell is shown in cellular level, MDV3100 drug resistance is overcome in drug resistant CRPC model, the progression of disease of castration-resistant prostate cancer can be significantly inhibited, application prospect with treatment prostate cancer.
Description
Technical field
The present invention relates to pharmaceutical technology fields, and in particular to small molecule compound QW07 treats prostatic disorders medicine in preparation
Application in object.
Background technique
Prostate cancer (Prostate Cancer, PCa) is that the most common urogenital system is pernicious swollen in middle-aging male
One of tumor.According to CACANCER J CLIN in 2016, prostate cancer disease incidence in American male occupy first, it is lethal
Rate occupies second, and the prostate-cancer incidence and the death rate in China increasingly rise, and serious harm health of people is serious, for forefront
The research of gland cancer also increasingly becomes hot spot.
At the initial stage that prostate cancer is made a definite diagnosis, the mode of " actively monitoring " is usually taken in treatment method.Causing symptom and is swelling
When tumor is also confined to prostatic, the treatment method of standard includes that prostate original position resection and radiotherapy etc. are locally controlled
Treatment method.But prostate cancer incubation period is longer, it is difficult to discover, when discovery often has developed into advanced prostate cancer.For evening
Phase prostate cancer, standard treatments are castration or Androgen deprivation therapy (Androgen Deprivation at present
Therapy, ADT), main includes operation castration and medical castration, but (18-48 is a for section in different times by most patients
Month) will recur, the situation that castration is resisted is presented, leads to castration-resistant prostate cancer (Castration Resistant
Prostate Cancer, CRPC), it shows to eventually lead to insensitive and to medical castration drug the tolerance of operation castration
It is dead.Very limited for the treatment method of CRPC at present, Food and Drug Adminstration of the US (FDA) approval is treated for CRPC
Drug only have conventional chemotherapeutic drugs Cabazitaxel and the second generation targeting AR drug --- androgen synthetic inhibitor Ah's bit
Dragon and the miscellaneous Shandong amine (MDV3100) of antiandrogen grace, but with the progress of disease, also will appear the drug resistance to said medicine, most
Lead to death eventually, i.e., is clinically in the state to past medical help at present for the CRPC patient in advanced stage.
Report according to market survey company Decision Resources claims, from the point of view of global 7 big drug markets, prostate
The market value of cancer drug rises to 5,700,000,000 dollars in 2014 from nearly 4,000,000,000 dollars of 2009, it is contemplated that will be into one by 2019
Step increases to 8,700,000,000 dollars.Since the age of onset of prostate cancer is usually later, thus patients show aging, and population
Aging already becomes worldwide trend, as being directed to clinical resistance or castration-resistant prostate cancer, targets androgen receptor N
Antiprostate cancer of new generation is developed at end, and studies its molecular mechanism, it will help the survival rate of patients with prostate cancer is improved,
Improve its quality of life
Therefore, in conjunction with the newest Basic Research Results of CRPC Forming Mechanism, exploitation overcomes drug resistance to treat the novel of CRPC
The drug of small-molecule drug, particularly the treatment CRPC with China's independent intellectual property right, and the molecular mechanism of its effect is illustrated,
It lays the foundation for subsequent new drug development, there is important theory significance and wide application prospect.
Summary of the invention
The invention proposes one kind QW07 as shown in formula (I) (European Journal of Medicinal
Chemistry 120 (2016) 13-25) small molecule compound or its hydrate or pharmaceutically acceptable salt be before preparation treatment
Application in the drug of column gland cancer disease;Formula (I) QW07 not only can treat early stage androgen-dependent prostate cancer,
It can treat advanced stage castration-resistant prostate cancer and metastatic prostate cancer, formula (I) QW07 is for inhibiting prostate cancer
Proliferation, growth, transfer, infiltration, Clone formation etc..
Wherein, QW07 shown in formula (I) is a kind of small molecule compound, molecular formula C26H33O4, molecular weight 423.55.
In present invention application, formula (I) QW07 inhibits the expression of overall length androgen receptor (AR-FL) downstream gene, or
Inhibit the expression of the androgen receptor downstream gene of missing ligand domain, or inhibits R1881 or Forskolin (FSK) induction
Androgen receptor downstream gene expression;The downstream gene includes PSA, FKBP5, SLC45A3 and TMPRSS2 etc..
In present invention application, formula (I) QW07 inhibits overall length androgen receptor in more plants of Prostatic cancer cell lines
(AR-FL) transcriptional activity of the androgen receptor (AR-Vs) of activity and missing ligand domain.In a specific embodiment,
The endogenous full-length androgen receptor AR-FL that formula (I) QW07 inhibits R1881 to induce in LNCaP and 22RV1 cell is living
Property;The exogenous overall length androgen receptor AR-FL transcriptional activity that formula (I) QW07 inhibits R1881 to induce in PC3 cell;
Formula (I) QW07 inhibits the transcriptional activity of the androgen receptor of exogenous missing ligand domain, the hero in PC3 cell
Hormone receptor includes AR-V7, AR-567esAnd AR-1-651.
The present invention application in, formula (I) QW07 can directly with AR-NTD ining conjunction with, block such as CBP (N-terminal corotation record because
Son) etc. altogether regulatory protein and AR-NTD interaction, inhibit androgen receptor transcription complex formation, the formula (I)
QW07 can also significantly block/inhibit androgen receptor AR and its target gene promoters and enhancer (androgen receptor bulk effect member
Part, ARE) combination, inhibit regulation of the androgen receptor to gene expression downstream.
In present invention application, formula (I) QW07 inhibits the proliferation of prostate gland cancer cell in vitro and/or in vivo.It is described
Prostate gland cancer cell includes the Prostatic cancer cell lines of the Prostatic cancer cell lines of androgen receptor feminine gender, the androgen receptor positive
And the Prostatic cancer cell lines of R1881 or Forskolin (FSK) induction;Wherein, the prostate of the androgen receptor feminine gender
Cancer cell line includes PC3, PC3M, DU145, PNT1A etc., and the Prostatic cancer cell lines of the androgen receptor positive include
LNCaP, VCaP, 22RV1, CHPC etc..Wherein, the Prostatic cancer cell lines relative to androgen receptor feminine gender, the formula (I)
QW07 becomes apparent the Prostatic cancer cell lines proliferation inhibiting effect of the androgen receptor positive.
Wherein, the 22RV1 cell strain and VCaP cell strain are Bicalutamide or the invalid androgen receptor of MDV3100 treatment
The cell strain of body mutation.
Wherein, the LNCaP is androgen-dependent prostate cancer cell line.
Wherein, the CHPC is primary isolated Prostatic cancer cell lines.
In present invention application, formula (I) QW07 is able to suppress the growth of Prostatic cancer cell lines;The prostate cancer is thin
Born of the same parents' strain includes LNCaP, 22RV1, VCaP, CHPC.
In present invention application, formula (I) QW07 can inhibit castration-resistant prostate cancer cell in mouse model
Strain growth, wherein the Prostatic cancer cell lines include Bicalutamide or MDV3100 treatment it is invalid androgen receptor mutation
Prostatic cancer cell lines 22RV1 and VCaP.
In present invention application, formula (I) QW07 inhibits the Clone formation of Prostatic cancer cell lines;Wherein, before described
Column adenocarcinoma cell strain includes 22RV1, LNCaP, VCaP, CHPC.
In present invention application, the cell cycle of formula (I) the QW07 retardance AR positive prostate cancer cells strain is without causing
Apoptosis;The cell cycle of formula (I) the QW07 retardance AR positive cell strain G0/G1 phase;Wherein, the AR positive cell
Strain is including 22RV1, LNCaP etc..
In present invention application, hydrate or pharmaceutically acceptable salt of formula (I) the QW07 compound etc. have and formula
(I) the identical inhibitory effect of QW07.
The invention also provides QW07 shown in formula (I) inhibit androgen receptor transcriptional activity in application and side
Method.
The invention also provides formula (I) QW07 inhibit in vitro and/or in vivo the proliferation of prostate gland cancer cell application and
Method.The prostate gland cancer cell includes the forefront of the Prostatic cancer cell lines of androgen receptor feminine gender, the androgen receptor positive
The Prostatic cancer cell lines of adenocarcinoma cell strain and R1881 induction;Wherein, the prostate gland cancer cell of the androgen receptor feminine gender
Strain including PC3, PC3M, DU145, PNT1A etc., Prostatic cancer cell lines LNCaP, VCaP of the androgen receptor positive,
22RV1, CHPC etc..Wherein, the Prostatic cancer cell lines relative to androgen receptor feminine gender, formula (I) QW07 is to androgen
The Prostatic cancer cell lines proliferation inhibiting effect of receptor positive becomes apparent.Particularly, formula (I) QW07 of the present invention can press down
The cell strain 22RV1 cell strain and VCaP cell strain of the invalid androgen receptor mutation of Bicalutamide or MDV3100 treatment processed increase
It grows.
The invention also provides QW07 shown in formula (I) to inhibit application and method in Prostatic cancer cell lines growth.
In the present invention, formula (I) QW07 can inhibit R1881 induce overall length androgen receptor (AR-FL) activity with
And the transcriptional activity of the androgen receptor (AR-Vs) of missing ligand domain.Formula (I) QW07 equally can inhibit prostate cancer thin
The growth of born of the same parents strain LNCaP, 22RV1, VCaP and the primary prostate cell strain in patient source.
The invention also provides formula (I) QW07 in the application and method for generating inhibiting effect to androgen receptor.
The present invention application in, formula (I) QW07 can be bound directly with AR, and inhibit AR transcription complex formation and its
And the combination of downstream gene promoter and enhancer also inhibits the transcriptional activity of overall length and truncated-type AR, shows in cellular level
Out to the selective depression of AR positive cell, and prostate cancer is significantly inhibited in drug resistant CRPC model, and overcome
The resistance problems of MDV3100.
The present invention screens to obtain small molecule compound formula (I) QW07, the compound energy by chemical-activated luciferase gene expression
The proliferation of enough strong inhibition prostate gland cancer cells in vitro and in vivo, and can in conjunction with the N-terminal structural domain of androgen receptor,
It significantly inhibits overall length androgen receptor and lacks the transcriptional activity of the androgen receptor of ligand domain.
The present invention is research shows that formula (I) QW07 can not only efficiently inhibit androgen-dependent prostate cancer cell line LNCaP
Proliferation, and (cell strain expresses the prominent of sustained activation to the Prostatic cancer cell lines 22RV1 that androgen receptor can be inhibited to be mutated
Become androgen receptor, and it is resistant to clinical medicine androgen receptor antagonists Bicalutamide and MDV3100) proliferation.This
Outside experiments have shown that the Prostatic cancer cell lines of expression androgen receptor have preferable sensibility, formula (I) QW07 to formula (I) QW07
The interaction of the total regulatory protein and AR-NTD such as CBP can be blocked, androgen receptor is inhibited to turn directly in conjunction with AR-NTD
The formation of complex is recorded, to block the combination of AR and downstream gene promoter and enhancer, inhibits downstream gene expression.Root
As a result, formula (I) QW07 not only can treat early stage androgen-dependent prostate cancer on accordingly, it also can treat advanced stage castration and support
Refractory prostate cancer and metastatic prostate cancer.
In present invention application, " castration-resistant prostate cancer (CRPC) " refers to that prostate cancer patient controls through castration
It treats (Testectomy or androgen deprivation reduce androgen) to recur afterwards, before the invalid prostate cancer of castration is known as castration-resistant
Column gland cancer, CRPC are the major reasons for leading to prostate cancer patient's death.
In present invention application, " androgen receptor (AR) " refers to, androgen receptor (AR) is the consideration convey of Ligand activation
The factor is recorded, AR albumen includes several functional areas: N-terminal regulatory region, the combined area DNA (DBD), hinge area and ligand-binding domain
(LBD).AR conformation can be changed after androgen is in conjunction with LBD, AR is separated with heat shock molecular chaperones (HSP), and new AR is compound
Object enters in core in conjunction with the target gene DNA androgen response factor, and activation AR downstream target gene includes current diagnosis prostate
The expression of the common index PSA of cancer and the genetic transcription of promoting growth of cell proliferation, the major transcription functional areas of AR are located at NTD
End, therefore lack LBD and will lead to the sustained activation of AR.In the present invention, " androgen receptor " includes endogenous and external source overall length
Androgen receptor (AR-FL) and truncated-type androgen receptor (androgen receptor of missing ligand domain) (AR-Vs), or
Overall length androgen receptor (AR-FL) activity of R1881 induction and the androgen receptor (AR-Vs) of missing ligand domain.
In present invention application, " the luciferase reporter gene detection (Luciferase assay) " refers to, fluorescein
Enzyme (Luciferase, LUC) Reporter System is to detect firefly luciferase so that fluorescein (luciferin) is substrate
A kind of (firefly luciferase) active reporting system.The present invention has constructed a screening and AR-NTD transcription is inhibited to live
Luciferase (Luciferase) reporter gene screening system of property, androgen receptor in conjunction with androgen after can star the matter
Grain gives expression to luciferase, and luciferase can be catalyzed its substrate luciferin and issue fluorescence, and the intensity by detecting fluorescence just can
Detect the degree of Androgen Receptor Activation;Renilla internal reference reporter gene has renilla luciferase, can express sea pansy fluorescein
Enzyme can determine whether the transfection efficiency of every group of cell is consistent by the plasmid, and androgen is to Renilla internal reference reporter gene matter
Grain does not influence.
The invention also provides a kind of pharmaceutical compositions, including formula (I) the QW07 compound or its hydrate or medicine
Acceptable salt and pharmaceutically acceptable carrier on.
The invention also provides formula (I) QW07 or its hydrate or pharmaceutically acceptable salt or pharmaceutically acceptable loads
Body and the composition for containing formula (I) QW07 are preparing the application in the drug for treating malignant tumour.Wherein, described pernicious swollen
Tumor be androgen receptor extremely expand and mutation malignant tumour, including prostate cancer, breast cancer, lung cancer, colon cancer, the cancer of the brain,
Cutaneum carcinoma, bladder cancer, kidney etc..
In the present invention, the present invention is by the 1-558 amino acids at the end androgen receptor NTD and Gal4DBD amalgamation and expression, simultaneously
Cotransfection contains the Gal4-luciferase plasmid of 5 × Gal4binding site, has constructed a screening and has inhibited AR-NTD
Luciferase (Luciferase) reporter gene screening system of transcriptional activity.This experiment is with existing small point of inventor laboratory
Sub- compound library is drug screening source, and screening to have obtained by above-mentioned Luciferase reporter gene has strong inhibition to AR-NTD
The small molecule compound QW07 of effect.Formula (I) QW07 can significantly inhibit endogenous and external source AR-FL and AR- under low concentration
The transcriptional activity of Vs and its expression regulation to downstream gene.In cellular level, formula (I) QW07 can selectively inhibit AR positive
Property cell strain proliferation and migration, either hormone-dependent type or independent form expression androgen receptor prostate cancer it is thin
Born of the same parents' strain.Subsequent results of animal shows that formula (I) QW07 can inhibit to concentration dependant the growth of CRPC, and is playing
Lower toxicity is shown under the drug concentration of effect.The treatment castration-resistant prostate cancer of newest F DA approval at present
The drug of (Castration Resistant Prostate Cancer, CRPC) include abiraterone (Abiraterone) and
MDV3100, both drugs are both for androgen receptor signal path, in the treatment side castration-resistant prostate cancer (CRPC)
Face obtains preferably certain curative effect, but after said medicine is treated, patient can still generate drug resistance, and above two
Medicine is not the drug of China's independent intellectual property right, and the new breakthrough that the present invention obtains in terms of prostate cancer therapy, is successfully filled up
The blank in the domestic field.
Detailed description of the invention
Fig. 1 show luciferase reporter gene and detects in each Prostatic cancer cell lines formula (I) QW07 to overall length androgen
The inhibitory effect of receptor active.Wherein, (A) indicates that formula (I) QW07 inhibits R1881 to be induced in prostate gland cancer cell LNCaP
Overall length androgen receptor (AR-FL) downstream gene expression;(B) indicate that formula (I) QW07 presses down in prostate gland cancer cell LNCaP
Overall length androgen receptor (AR-FL) transcriptional activity that R1881 processed is induced;(C) indicate formula (I) QW07 in prostate gland cancer cell
The transcription of the overall length androgen receptor (AR-FL) and truncated-type androgen receptor (AR-Vs) that inhibit R1881 to be induced in 22RV1
Activity;(D) formula (I) QW07 suppression in prostate gland cancer cell PC3 (having transfected external source wild type full-length androgen receptor AR) is indicated
Overall length androgen receptor (AR-FL) transcriptional activity that R1881 processed is induced.
Fig. 2 show formula (I) QW07 in each Prostatic cancer cell lines of luciferase reporter gene detection and swashs to truncated-type hero
The active inhibitory effect of plain receptor (AR-Vs).Wherein, (A) indicates that formula (I) QW07 inhibits in prostate gland cancer cell 22RV1
The overall length androgen receptor (AR-FL) and truncated-type androgen receptor (AR-Vs) downstream gene that Forskolin (FSK) is induced
Expression;(B) formula (I) QW07 suppression in prostate gland cancer cell PC3 (having transfected external source wild-type androgen receptor AR-V7) is indicated
Androgen receptor (AR-V7) transcriptional activity processed;(C) indicate that formula (I) QW07 (it is wild to have transfected external source in prostate gland cancer cell PC3
Type androgen receptor AR-567es) in inhibit androgen receptor (AR-567es) transcriptional activity;(D) indicate formula (I) QW07 in forefront
Inhibit androgen receptor (AR-1-651) transcription in adenocarcinoma cell PC3 (having transfected external source wild-type androgen receptor AR-1-651)
Activity.
Fig. 3 show formula (I) QW07 and can combine with AR, and binding site is located at the N-terminal structural domain of androgen receptor
(AR-NTD).Wherein, (A) indicates that compound MDV3100 can be combined in a manner of concentration dependant with AR-FL;(B) expression
(I) QW07 can be combined in a manner of concentration dependant with AR-FL;(C) indicate compound MDV3100 cannot in vitro with AR-17H
It combines;(D) indicate that compound EPI001 can be combined with AR-17H in vitro;(E) indicate formula (I) QW07 can in vitro with
AR-17H is combined.
Fig. 4 show formula (I) QW07 to the inhibitory effect of each Prostatic cancer cell lines proliferation activity.Wherein, (A) expression
(I) proliferation of QW07 selective depression androgen receptor (AR) positive prostate cancer cells;(B) indicate that formula (I) QW07 inhibits
The prostate gland cancer cell proliferation activity that R1881 is induced (wherein, MDV refers to MDV3100);(C) formula (I) QW07 is shown to before each
The inhibitory effect and its statistical result that column adenocarcinoma cell strain clone is formed.
Fig. 5 show influence of formula (I) QW07 to prostate gland cancer cell LNCaP, 22RV1 cell cycle distribution and apoptosis.
Wherein, (A) indicates the cell cycle of formula (I) QW07 retardance prostate gland cancer cell LNCaP, 22RV1;(B) formula (I) QW07 is indicated not
The apoptosis of obvious induction prostate gland cancer cell LNCaP, 22RV1.
Fig. 6 show formula (I) QW07 in vivo to the inhibitory effect of Prostatic cancer cell lines 22RV1 growth.Wherein, (A)
Indicate different disposal group 22RV1 tumour white light figure;(B) statistical result of different disposal group 22RV1 tumor weight is indicated;(C) table
Show the change curve of different disposal group gross tumor volume;(D) different disposal group mouse weight change curve is indicated;(E) it show not
With processing group nude mice viscera tissue H&E coloration result.
Fig. 7 show formula (I) QW07 in vivo to the inhibitory effect of Prostatic cancer cell lines VCaP growth.Wherein, (A) table
Show different disposal group VCaP tumour white light figure;(B) statistical result of different disposal group VCaP tumor weight is indicated;(C) it indicates not
With the change curve of processing group gross tumor volume;(D) different disposal group mouse weight change curve is indicated;(E) it show and does not exist together
The expression of AR downstream gene PSA and TMPRSS2 in reason group tumor tissues.
Fig. 8 show the mechanism of action that formula (I) QW07 inhibits androgen receptor transcription.(A) it show formula (I) QW07 inhibition
AR and its N-terminal are total to the combination of transcription factor CBP;(B) inhibit AR and its in Prostatic cancer cell lines LNCaP for formula (I) QW07
The combination of target gene promoters and enhancer;(C) inhibit AR and its target base in Prostatic cancer cell lines VCaP for formula (I) QW07
Because of the combination of promoter and enhancer.
Specific embodiment
In conjunction with following specific embodiments and attached drawing, the present invention is described in further detail, protection content of the invention
It is not limited to following embodiment.Without departing from the spirit and scope of the invention, those skilled in the art it is conceivable that change
Change and advantage is all included in the present invention, and using appended claims as protection scope.Implement process of the invention,
Condition, reagent, experimental method etc. are among the general principles and common general knowledge in the art in addition to what is specifically mentioned below,
There are no special restrictions to content by the present invention.
Embodiment 1: formula (I) QW07 inhibits the transcriptional activity of androgen receptor in vitro
Technical method:
1, the culture of cell: cell used is purchased from Chinese Academy of Sciences Shanghai cell bank in this experiment, and part cell comes from Shanghai City
Regulate and control biology key lab Weng Jiemin and teaches laboratory;Separately there is one plant of cell to cure from the attached Changhai of The 2nd Army Medical College
Institute is from the primary isolated prostate gland cancer cell of prostate cancer tissue of patient, and cell culture is in 37 DEG C of constant incubator (humidity
95%, CO2 concentration 5%) in, it is incubated in the culture medium of the RPMI-1640 containing 10% fetal calf serum (Front) (Gibco).
2, luciferase reporter gene detects: testing plasmid used is AR (1-558)-Gal4-DBD and Gal4-
Luciferase and Renilla (internal reference reporter gene).AR (1-558)-Gal4-DBD is by 1-558 ammonia of androgen receptor N-terminal
Base acid (AR-NTD) and the end yeast transcription factor GAL4N 1-147 amino acids (Gal4-DBD) are connected to express into pcDNA3.1 and be carried
Body is built-up, being capable of amalgamation and expression androgen receptor NTD and Gal4-DBD after being transferred to eukaryocyte.Gal4-
5 × Gal4binding site is had on luciferase plasmid, specifically can be identified and be combined by Gal4-DBD.When melting
After hop protein AR (1-558)-Gal4-DBD is expressed, can under the traction of Gal4-DBD, specifically with Gal4-
Binding site is combined, and the expression of downstream luciferase is originated due to transcriptional activity that AR-NTD has, and fluorescein
Enzyme can be catalyzed its substrate luciferin and issue fluorescence, and the intensity by detecting fluorescence just can detect the journey of Androgen Receptor Activation
Degree;Renilla internal reference reporter gene has renilla luciferase, can express renilla luciferase, can be determined by the plasmid
Whether the transfection efficiency of every group of cell is consistent.
Experimental result:
As shown in Fig. 1 (B), Fig. 1 (C), Fig. 1 (D), Fig. 2 (B), Fig. 2 (C), Fig. 2 (D), dosage of formula (I) QW07 at 10 μM
Under, it can reduce the overall length androgen receptor (AR-FL) and truncated-type androgen that R1881 is induced in different Prostatic cancer cell lines
The transcriptional activity of receptor (AR-Vs), it was demonstrated that the overall length androgen receptor that formula (I) QW07 can not only inhibit R1881 to induce in vitro
Transcriptional activity, truncated-type androgen receptor transcription can also be inhibited active;In addition, the formula (I) as shown in Fig. 1 (A) and Fig. 2 (A)
The expression for the androgen receptor downstream gene that QW07 can not only inhibit R1881 or Forskolin (FSK) to induce, can also press down
The expression of truncated-type androgen receptor (the saltant type androgen receptor of sustained activation) downstream gene processed, the downstream gene include
PSA, FKBP5, SLC45A3 and TMPRSS2.Wherein Forskolin (FSK) is a kind of common adenyl cyclase activator,
The activity of cAMP dependent kinases (PKA) approach excitement AR-NTD can be passed through.
Embodiment 2: formula (I) QW07 and positive compound (MDV3100) can be combined with AR
Technical method:
Surface plasma resonance technology (Surface Plasmon Resonance technology, SPR) is based on SPR
The cutting edge technology for detecting ligand and analyte effect on bio-sensing chip (Biosensor chip), compared with traditional means,
SPR has outstanding advantages of being not necessarily to that sample is marked, energy real-time monitoring, high sensitivity.The present invention buys commercialized AR-
NTD (1-558 amino acids) albumen, then carry out SPR detection.Operation is summarized as follows: by induction chip insertion Bicore T200 inspection
Examining system, with PBS-T buffer balance system.Testing protein is dissolved in NaAc, then passes through amino for above-mentioned proteopexy
On the individual vertical access of induction chip, grope relevant parameter, after radix is stablized, is dissolved in the difference of PBS-T buffer
The QW07 level of concentration flows into chip, and experimental result data is with software by carrying out dynamic analysis.
Experimental result:
As shown in Fig. 3 (A) and Fig. 3 (B), formula (I) QW07 and positive compound (MDV3100) can be with the shapes of concentration dependant
Formula is combined with AR.And in the Binding experiment with AR-17H, formula (I) QW07 similarly can be in combination, and positive chemical combination
Object EPI001 also can be in combination.And binding site is located at AR-LBD, it can be in small molecule compound of the extremely low concentration in conjunction with AR
For MDV3100 no longer in conjunction with AR-17H, experimental result such as Fig. 3 (C), Fig. 3 (D) and Fig. 3 (E) are shown.Above the experimental results showed that,
Similarly with EPI001, formula (I) QW07 can be combined with AR in vitro, and binding site is located at the N-terminal structural domain of androgen receptor
(AR-NTD)。
Embodiment 3: formula (I) QW07 is to the proliferation activity of different prostate gland cancer cells and the inhibiting effect of Clone formation
Technical method:
1, SRB (Sulforhodamine) method measures cell Proliferation: different prostate cell strains are with 5 × 103A/hole density connects
Kind to 96 orifice plates (Corning), for 24 hours after, this monomeric compound of various concentration formula (I) QW07 is added, equivalent is added in control group
DMSO, each group set 6 multiple holes.Continue culture respectively for 24 hours, after 48h and 72h, add precooling TCA (trichloroacetic acid, 50%, w/
V) 4 DEG C of incubation 60min or more fix cell.After fixation, flowing water is rinsed 5 times, is air-dried.50 μ l SRB dye liquors of every hole addition (4%,
W/V), incubation at room temperature 10min dyeing.Dye liquor is sucked out, every hole is added 1% acetic acid, 100 μ l and washes 5 times, removes unbonded dyestuff.Wind
After dry, it is 100 μ l of 10mMTris solution, the SRB dyestuff that concussion dissolution combines that concentration, which is added, in every hole.96 orifice plates are placed in microplate reader
In (SPECTRAMAX 190), OD value is measured under 515nm wavelength.Statistically analyze influence of the drug for cell proliferation level.
2, colony formation: 1000 prostate gland cancer cells are linked into 6 orifice plates, Fig. 4 is added after cell is adherent
Shown in concentration formula (I) QW07, the cells are fixed with paraformaldehyde after a week, then uses violet staining, takes pictures and count
Number is cloned, every group of experiment is in triplicate.Clone formation is one of the effective ways for detecting cancer cell multiplication ability.
Experimental result:
As shown in figure 4, formula (I) QW07 has significant inhibitory effect to the proliferation of each strain prostate gland cancer cell.Also,
Formula (I) QW07 is other than significant to common Prostatic cancer cell lines proliferation inhibiting effect, to from the primary separation of clinical patient
Prostatic cancer cell lines also have effect same.The experimental result as shown in Fig. 4 (C), also strong inhibition is each simultaneously by formula (I) QW07
The Clone formation of strain prostate gland cancer cell.Using above-mentioned cell proliferation experiment and colony formation, inventor compares formula
(I) sensibility of cell strain proliferation QW07 positive to androgen and negative, experimental result such as Fig. 4 (A), Fig. 4 (B) and Fig. 4 (C)
It is shown, whether in terms of cell Proliferation or Clone formation, prostate gland cancer cell of formula (I) QW07 to the androgen receptor positive
Strain inhibitory effect becomes apparent.The above experiment shows that formula (I) QW07 can significantly inhibit each strain prostate gland cancer cell in low concentration
Proliferation, and it is more significant to the prostate gland cancer cell inhibitory effect of the androgen receptor positive.
Embodiment 4: formula (I) QW07 is to the Cycle Arrest of prostate gland cancer cell and the influence of Apoptosis
Technical method:
1, flow cytomery Apoptosis-Annexin V/PI double-staining: in normal cell, phosphatidyl silk
Propylhomoserin (Phosphatidylserine, PS) is located at the inside of cell membrane, but early stage Apoptosis, and PS can be from cell membrane
The surface for turning inside out cell membrane, is exposed in extracellular environment.Annexin-V is a kind of Ca2+ that molecular weight is 35.8KD
Dependence cardiolipin binding protein can be specifically bound with PS high-affinity.Using AnnexinV-FITC as fluorescence probe, benefit
It can detect the generation of Apoptosis with flow cytometer or fluorescence microscope.Propidium iodide (Propidine Iodide, PI) is
A kind of nucleic acid dye, it cannot penetrate complete cell membrane, but in the cell and dead cell of apoptosis middle and advanced stage, PI can be through thin
After birth and make the red dye of nucleus.Therefore Annexin-V is matched into use with PI, so that it may by the cell in apoptosis early advanced stage and extremely
Cell differentiation comes, and can detecte the ratio of apoptotic cell by flow cytometer.
2, the cell cycle tests: can be in conjunction with fluorescent dye such as the binding characteristic of PI, different times using intracellular DNA
Due to DNA content difference to which the amount of the fluorescent dye combined is also different, using flow cytomery fluorescence intensity not yet
Together, thus reacting cells cycle stage.Specific steps are as follows: the prostate gland cancer cell of logarithmic phase is taken to be inoculated in 6cm ware,
For 24 hours afterwards be cell it is adherent after be added do not have to concentration compound, after drug-treated for 24 hours after, cell is collected by centrifugation, is cleaned with PBS
Twice of cell, after the PBS for centrifuge tube remnants that exhaust, the fixed cell of 75% ethyl alcohol of 100 μ L is added in every pipe, carefully with liquid-transfering gun piping and druming
It is resuspended sufficiently in born of the same parents, and 4 DEG C overnight.2nd day, cell is collected by centrifugation, gently outwells alcohol, cell centrifugation is resuspended with 1mL PBS
Cleaning 2 times, room temperature is protected from light item incubation 30 after 1 μ LRNA enzyme (10mg/mL) and 5 μ L PI (5mg/mL) mixing are added under the conditions of being protected from light
Minute, it is then transferred to after the streaming pipe of 5mL i.e. with the flow cytomery cell cycle.Count the cell content in each period.
Experimental result:
As shown in Fig. 5 (A) and Fig. 5 (B), formula (I) QW07 induces prostate gland cancer cell (LNCaP and 22RV1) the G0/G1 phase
Cell-cycle arrest, and to Apoptosis without be obviously promoted effect.
Embodiment 5: anti-tumor activity of formula (I) QW07 in castration-resistant prostate cancer (CRPC) mouse model
Technical method:
Two Prostate Carcinoma of Mice growth models are established with Human Prostate Cancer Cells 22RV1 and LNCaP respectively.By 5,000,000
A Human Prostate Cancer Cells 22RV1 cell and LNCaP cell subcutaneous injection are to immunodeficient mouse (BLAB/c-nude, nude mice)
Dorsal sc grows to 100mm to subcutaneous tumor3When left and right, by mouse be divided into three groups (mean tumour volume is identical,
Formula (I) QW07 that 2mg/kg is dissolved in DMSO is injected intraperitoneally in dosing group mouse daily, and positive drug group injects 10mg/kg Bicalutamide
(Bicalutamide, BIL), control group only inject DMSO, measure and record the length and width of mouse weight and tumour daily,
22RV1 tumour (n >=7) and LNCaP tumour (n >=4) put to death mouse after being administered 24 days and 49 days respectively, take subcutaneous tumor, take pictures,
It is calculated according to formula volume=length × wide 2 × 0.52, counts gross tumor volume.
Experimental result:
As shown in Figure 6 and Figure 7, formula (I) QW07 significantly inhibits effect to CRPC tumour growth in CRPC animal model,
And inhibitory effect is better than positive drug MDV3100.As a result such as Fig. 6 (A), Fig. 6 (B), Fig. 6 (C), Fig. 7 (A), Fig. 7 (B) and Fig. 7 (C)
Shown, formula (I) QW07 can overcome drug resistance, hence it is evident that inhibit the growth of tumour, no matter in tumor size or tumor quality
Significant difference all is presented with control group and MDV3100 group, and also there is the concentration dependent to QW07 simultaneously.Especially in VCaP-
In CRPC model, the QW07 of high dose shows similar inhibition tumour growth effectiveness with the EPI001 with dosage.By small
Mouse changes of weight and viscera tissue slice H&E dyeing show that the QW07 of effective dose not will cause apparent histoorgan damage
Wound, as a result as shown in Fig. 6 (D), Fig. 6 (E) and 7 (D).In addition, experimental result is as schemed by detection AR downstream gene expression situation
Shown in 7 (E), compared with the control group, the expression of MDV3100 processing group AR downstream gene PSA and TMPRSS2 without significant change, and
There is apparent downward in the gene expression of EPI001 processing group and QW07 processing group, and further having proved QW07 may be mainly
By inhibiting AR signal path to realize the inhibition to CRPC tumour growth.
In conclusion formula (I) QW07 can inhibit the growth of castration resistance prostate cancer in mouse model.
Embodiment 6: formula (I) QW07 is able to suppress androgen receptor transcription, to inhibit downstream gene expression
Technical method:
1, immunoblotting (western blot) is tested: after adding various concentration drug-treated, cell mentions cell through cracking
Albumen is taken, albumen is separated by electrophoresis protein example with polyacrylamide gel PAGE after denatured by boiling, is then transferred into nitric acid
On cellophane, it is incubated for two hours with the antibody (primary antibody) of the antibody of androgen receptor AR and other albumen first, then use band
There is two antibody incubation of fluorescent marker one hour, finally with the expression for sweeping film instrument Odyssey and detecting the albumen.
2, polymerase chain reaction (Polymerase Chain Reaction, PCR) is tested: cell is through various concentration medicine
After object processing, cDNA is obtained after reverse transcription with TRIzol separation and Extraction RNA, RNA, with different AR downstream specific primers
(such as PSA, TMPRSS2) detects the expression of its mRNA by real-time quantitative PCR (Q-PCR).
3, co-immunoprecipitation (Co-Immunoprecipitation): co-immunoprecipitation (Co-
Immunoprecipitation based on being) specificity effect between antibody and antigen for study protein mutual
The classical way of effect.It is to determine two kinds of protein effective ways that physiological interacts in intact cell.Its principle is:
Interaction quilt when cell is cleaved under the conditions of non denatured, in intact cell between existing many protein-proteins
It keeps down.If precipitating X with the antibody mediated immunity of protein X, can also be precipitated down with the protein Y that X is combined in vivo
Come.The proreinA of multi-purpose purification is combined in advance at present is solidificated on the beads of agarose, is allowed to and the solution containing antigen
And after antibody response, the proreinA energy adsorption antigen on beads achievees the purpose that purification.This method is usually used in measurement two
Whether kind target protein combines in vivo.
4, shape will chromatin immune co-precipitation (Chromatin Immunoprecipitation Assay, CHIP): be grown
After the good cell of state is digested, counted, appropriate access in 15cm ware is cultivated.After cell is adherent, filtered with containing 5% active carbon
Serum without phenol red medium starved cells 12 hours.Original culture medium is absorbed, be added comprising R1881 or FSK and includes simultaneously
The DMSO of equivalent is added in the culture medium of R1881 or FSK and various concentration untested compound, control group.After processing setting duration, add
Enter final concentration of 1% formaldehyde crosslinking cell 10 minutes, glycine is then added and terminates crosslinking.Collection is resuspended in cell pyrolysis liquid
Cell is simultaneously cracked on ice, and nuclease digestion DNA is then added, and the ultrasonic sample in ultrasonic water bath pot simultaneously, by long chain DNA
It is broken into the DNA fragmentation of 200-1000bp, becomes clear, centrifuging and taking supernatant to cell suspension.Specificity is added in supernatant
Primary antibody or control IgG, 4 DEG C of rotation are incubated overnight.After addition ProteinA/G beads rotation is incubated for 4 hours within second day, respectively
It is repeatedly washed with dilution buffer, low salt solutions, high level salt solution to reduce non-specific binding.Connection, albumen are made friends in 65 DEG C of water-baths
After enzyme K digests 2 hours, by the mating DNA Purification Kit of eluted product, purified product is obtained.Then sample is carried out glimmering
Fluorescent Quantitative PCR analysis.
Experimental result:
As shown in Fig. 8 (A), after formula (I) QW07 combination AR-NTD, hence it is evident that inhibit AR important regulatory protein CBP and AR- altogether
The interaction of NTD;Further experiment discovery, formula (I) QW07 significantly inhibit androgen receptor and its in prostate gland cancer cell
The combination of target gene promoters and enhancer (androgen receptor response element, ARE), and then inhibit the table of AR downstream gene
It reaches, as a result as shown in Fig. 8 (B) and Fig. 8 (C).
For above-described embodiment only for illustrating technical concepts and features of the invention, its object is to allow those skilled in the art
It cans understand the content of the present invention and implement it accordingly, it is not intended to limit the scope of the present invention.It is all according to the present invention
Equivalent change or modification made by the essence of content should all cover in the scope of the present invention.
Claims (16)
1. QW07 small molecule compound shown in formula (I) or its hydrate or pharmaceutically acceptable salt treat androgen in preparation
Application in receptor mutation and the abnormal drug for expanding relevant malignancy disease;
2. application as described in claim 1, which is characterized in that the malignant tumour include prostate cancer, breast cancer, lung cancer,
Colon cancer, the cancer of the brain, cutaneum carcinoma, bladder cancer, kidney.
3. application as claimed in claim 2, which is characterized in that formula (I) QW07 is used to inhibit proliferation, the life of prostate cancer
Long, transfer, infiltration, Clone formation.
4. application as claimed in claim 3, which is characterized in that formula (I) QW07 inhibits the forefront of androgen receptor feminine gender
Adenocarcinoma cell strain, the Prostatic cancer cell lines of the androgen receptor positive and R1881 or the prostate cancer of Forskolin induction are thin
The proliferation of born of the same parents' strain;Wherein, the Prostatic cancer cell lines of the androgen receptor feminine gender include PC3, PC3M, DU145, PNT1A, institute
The Prostatic cancer cell lines for stating the androgen receptor positive include LNCaP, VCaP, 22RV1, CHPC.
5. application as claimed in claim 3, which is characterized in that formula (I) QW07 for inhibit LNCaP, 22RV1, VCaP,
The growth of CHPC Prostatic cancer cell lines.
6. application as claimed in claim 3, which is characterized in that formula (I) QW07 inhibits castration-resistant prostate cancer thin
The growth of born of the same parents' strain.
7. application as claimed in claim 3, which is characterized in that formula (I) QW07 inhibits 22RV1, LNCaP, VCaP, CHPC
The Clone formation of Prostatic cancer cell lines.
8. application as claimed in claim 3, which is characterized in that formula (I) QW07 blocks the strain of AR positive prostate cancer cells
Cell cycle.
9. application as described in claim 1, which is characterized in that formula (I) the QW07 inhibition overall length androgen receptor AR-FL,
The androgen receptor of androgen receptor AR-Vs or R1881 or Forskolin induction of ligand domain is lacked to gene downstream
The regulation of expression.
10. application as described in claim 1, which is characterized in that formula (I) QW07 inhibits overall length androgen receptor AR-FL
With the transcriptional activity of the androgen receptor AR-Vs of missing ligand domain.
11. application as described in claim 1, which is characterized in that formula (I) QW07 inhibits the overall length hero of R1881 induction to swash
The androgen receptor AR-Vs transcriptional activity of plain receptor AR-FL activity and missing ligand domain.
12. application as claimed in claim 11, which is characterized in that formula (I) QW07 presses down in LNCaP and 22RV1 cell
The endogenous full-length androgen receptor AR-FL activity of R1881 induction processed and the external source for inhibiting R1881 induction in PC3 cell
Property overall length androgen receptor AR-FL transcriptional activity.
13. application as claimed in claim 11, which is characterized in that formula (I) QW07 inhibits exogenous in PC3 cell and lacks
The transcriptional activity of the androgen receptor of mismatch binding domains, the androgen receptor include AR-V7, AR-567esAnd AR-1-651.
14. a kind of pharmaceutical composition, which is characterized in that it include formula as described in claim 1 (I) QW07 compound or its
Hydrate or pharmaceutically acceptable salt and pharmaceutically acceptable carrier.
15. formula (I) QW07 compound as described in claim 1 or its hydrate or pharmaceutically acceptable salt, or as weighed
Benefit require 14 described in pharmaceutical composition in preparation treatment and androgen receptor mutation and the relevant malignant tumour disease of amplification extremely
Application in the drug of disease.
16. application as claimed in claim 15, which is characterized in that the malignant tumour is androgen receptor amplification extremely and dashes forward
Become malignant tumour, including prostate cancer, breast cancer, lung cancer, colon cancer, the cancer of the brain, cutaneum carcinoma, bladder cancer, kidney.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710859779 | 2017-09-21 | ||
CN2017108597796 | 2017-09-21 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN109528700A true CN109528700A (en) | 2019-03-29 |
Family
ID=65830668
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201711123382.7A Pending CN109528700A (en) | 2017-09-21 | 2017-11-14 | Application of the small molecule compound QW07 in preparation treatment prostatic disorders drug |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109528700A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110606814A (en) * | 2019-08-30 | 2019-12-24 | 华东师范大学 | Application of small molecule compound QW24 in preparation of medicine for treating colorectal cancer diseases |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104557602A (en) * | 2015-01-07 | 2015-04-29 | 华东师范大学 | Tricyclic diterpene derivative, as well as preparation method and application thereof in preparation of anti-tumour drug |
CN106928147A (en) * | 2017-03-14 | 2017-07-07 | 华东师范大学 | Tricyclic diterpene analog and preparation method thereof and its application in antiprostate cancer is prepared |
-
2017
- 2017-11-14 CN CN201711123382.7A patent/CN109528700A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104557602A (en) * | 2015-01-07 | 2015-04-29 | 华东师范大学 | Tricyclic diterpene derivative, as well as preparation method and application thereof in preparation of anti-tumour drug |
CN106928147A (en) * | 2017-03-14 | 2017-07-07 | 华东师范大学 | Tricyclic diterpene analog and preparation method thereof and its application in antiprostate cancer is prepared |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110606814A (en) * | 2019-08-30 | 2019-12-24 | 华东师范大学 | Application of small molecule compound QW24 in preparation of medicine for treating colorectal cancer diseases |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107012203B (en) | Application of PDCD4 as therapeutic target of antidepressant and/or anxiolytic drugs | |
CN102124335B (en) | Tumor suppressor-based susceptibility of hyperproliferative cells to oncolytic viral therapy | |
CN101632833B (en) | Prostatic cancer related gene and application thereof | |
Thies et al. | Stromal platelet–derived growth factor receptor-β signaling promotes breast cancer metastasis in the brain | |
CN105979780A (en) | Glucocorticoid inhibitors for treatment of prostate cancer | |
Gan et al. | The kynurenine derivative 3-HAA sensitizes hepatocellular carcinoma to sorafenib by upregulating phosphatases | |
CN104758292B (en) | PD-0332991 is preparing the purposes of prevention drug-resistant tumor drug | |
CN105078969B (en) | Application of the ailanthinone in the medicine for preparing treatment prostatic disorders | |
CN106047880A (en) | PVT1 siRNA-1055 for inhibiting blood tumor cell proliferation and application thereof | |
Zhang et al. | Effects of thalidomide on angiogenesis and tumor growth and metastasis of human hepatocellular carcinoma in nude mice | |
Cao et al. | Establishment of a novel mouse hepatocellular carcinoma model for dynamic monitoring of tumor development by bioluminescence imaging | |
CN109568299A (en) | Ambroxol purposes in preparing tumor chemotherapeutic drug Synergistic preparations | |
Bozzi et al. | High CD133 expression levels in gastrointestinal stromal tumors | |
CN107488733B (en) | Application of the miR-133b in prostate cancer with osseous metastasis diagnoses, predicts, treats | |
Tan et al. | Erchen Plus Huiyanzhuyu Decoction Inhibits the Growth of Laryngeal Carcinoma in a Mouse Model of Phlegm‐Coagulation‐Blood‐Stasis Syndrome via the STAT3/Cyclin D1 Pathway | |
CN109528700A (en) | Application of the small molecule compound QW07 in preparation treatment prostatic disorders drug | |
CN105063196B (en) | Proteasome inhibitor combines the application in cholangiocarcinoma treatment with cell autophagy activator | |
CN105624325A (en) | Marker for diagnosing and treating lung adenocarcinoma | |
CN109966479A (en) | Application of the EZH2 in preparation prevention or treatment polycystic kidney disease drug | |
CN104083368A (en) | Application of G-1 in preparation of G protein coupled receptor 30-based triple negative breast cancer targeting drugs | |
CN105247371A (en) | Treatment of insulin resistance through inhibitors of transcription factor TSC22D4 | |
CN103468799B (en) | EIF5A2 application in preparation treatment esophageal squamous cell carcinoma preparation | |
CN102099038A (en) | Materials and methods for suppressing and/or treating neurofibroma and related tumors | |
CN104662028B (en) | Compositions and methods for treating cancer with aberrant lipogenic signaling | |
KR102277435B1 (en) | Compositions for treating sorafenib-resistant liver cancer and Methods for providing information on treating sorafenib-resistant liver cancer |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190329 |
|
WD01 | Invention patent application deemed withdrawn after publication |