CN104825455B - The purposes of Buddhist nun is replaced according to Shandong - Google Patents
The purposes of Buddhist nun is replaced according to Shandong Download PDFInfo
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- CN104825455B CN104825455B CN201410258423.3A CN201410258423A CN104825455B CN 104825455 B CN104825455 B CN 104825455B CN 201410258423 A CN201410258423 A CN 201410258423A CN 104825455 B CN104825455 B CN 104825455B
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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
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
Abstract
The invention discloses a kind of new application that Buddhist nun (PCI 32765) is replaced according to Shandong, specifically, present invention discover that the non-small cell lung cancer for carrying EGFR T790M mutation and/or EGFR L858R mutation and/or EGFR delE746_A750 mutation can be treated for Buddhist nun according to Shandong, the non-small cell lung cancer of the double mutation of EGFR L858R and EGFR T790M is particularly carried.
Description
Technical field
The present invention relates to field of medicaments, a kind of new application of Buddhist nun (PCI-32765) is more particularly to replaced according to Shandong.
Background technology
Lung cancer is clinical common pulmonary malignant tumour, is generally divided into ED-SCLC (Small Cell Lung
Cancer, SCLC) and non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) two major classes.The morbidity of lung cancer
First of rate and the death rate row malignant tumour, wherein non-small cell lung cancer accounts for the overwhelming majority, the most generally.Non-small cell lung cancer exists
The global incidence of disease rises year by year, seriously threatens human health.Non-small cell lung cancer is the U.S., Japan and Western European countries' cancer
The lethal main cause of disease.For patients with terminal, although chemotherapy improves survival rate, chemotherapeutic to a certain extent
Thing also has significant toxicity to human body, thus is highly desirable to and can specifically target controlling for the key gene relevant with tumour growth
Treat agent (Schiller JH etc., N.Engl.J.Med., 346:92-98,2002).
EGF-R ELISA (Epidermal Growth Factor Receptor, EGFR) is that growth promotion is carcinogenic
Gene erbB or ErbBl protein, it is one of the ERBB families of family's proto-oncogene (proto-oncogenes)
Member, it is believed that played a crucial role in the occurrence and development of many cancers of the mankind.Especially, lung cancer, breast cancer, carcinoma of urinary bladder,
The enhanced expression of EGFR is observed in the cancer of the brain, neck cancer and stomach cancer and spongioblastoma.The ERBB families coding of oncogene
Related transmembrane receptor in four kinds of structures, i.e. EGFR, HER-2/neu (erbB2), HER-3 (erbB3) and HER-4 (erbB4).
Clinically, the amplification of ERBB oncogene and/or acceptor overexpression and palindromia and the patient in display tumour are had been reported
Prognosis difference correlation, and (L.Harris etc., 1999, Int.J.Biol.Markers, 14 related to the response of therapy:8-15；
J.Mendelsohn and J.Baselga, 2000, Oncogene, 19:6550-6565).
At present, by target spot of EGFR (for example (Gefitinib, Ji Fei are replaced Iressa tyrosine kinase inhibitor (TKI)
Buddhist nun) and Erlotinib (Erlotinib, Erlotinib)) etc. medicine obtained in the clinical treatment of non-small cell lung cancer it is huge into
Work(.However, receiving the patient of TKI inhibitor for treating often due to forming TKI drug resistances and facing the predicament of recurrence.The second generation
The non-reversible inhibitor of EGFR such as Canertinib (Canertinib), Afatinib (Afatinib), HKI-272
(Neratinib), pelitinib (Pelitinib) etc. has been enter into clinical test, but selection of these molecules to EGFR mutant
Property is poor, causes the clinical tolerance dosage of medicine relatively low.As a result, under maximum tolerated dose, medicine can not reach effectively in vivo
Concentration, thus it is invalid to most resistance patients.
In addition, it was found that in the non-small cell lung cancer using Gefitinib or erlotinib treatment EGFR-TKI responsive types
(NSCLC) when, often there is secondary resistance after 6~12 months in patient, wherein about 50% includes what is encoded by extron 20
T790M is mutated.Research thinks that T790M mutation hinder EGFR and EGFR micromolecular inhibitors (such as Gefitinib and Erlotinib)
Combination or increase EGFR and ATP affinity, thus cause resistance (Yun CH et al., Proc Natl Acad Sci
U S A.2008Feb 12；105(6):2070-5).
Buddhist nun (Ibrutinib, also referred to as PCI-32765) is replaced to be a kind of bruton's tyrosine kinase (Bruton's according to Shandong
Tyrosine kinase, Btk) inhibitor, it can be by way of being used alone or using with other therapeutic agents
Include lymthoma and inflammatory disease for treating autoimmune disease or illness, heteroimmunity disease or illness, cancer
Or illness.At present, it yet there are no on carrying EGFR T790M mutation and/or EGFR using according to Shandong for Buddhist nun (PCI-32765) treatment
The relevant report for the drug resistance Patients with Non-small-cell Lung that L858R is mutated and/or EGFR delE746_A750 are mutated.
The content of the invention
In view of foregoing technical problem, inventor conducts extensive research.As a result, inventor is it was unexpectedly observed that according to Shandong
For this bruton's tyrosine kinase inhibitor of Buddhist nun (PCI-32765) can effectively treat carrying EGFR L858R mutation and/or
The drug resistance non-small cell lung cancer that EGFR T790M are mutated and/or EGFR delE746_A750 are mutated, particularly treatment are carried
The drug resistance non-small cell lung cancer of the double mutation of EGFR L858R/T790M.More specifically, inventor has found, can be by using according to Shandong
Suppress the propagation (IC50 of the lung carcinoma cell of the foregoing mutation type of carrying various combination for Buddhist nun<1 μM), promote Apoptosis simultaneously
Suppress the size and number of cell colony, and for carrying EGFR WT (wild type) normal cell, Buddhist nun (PCI- is replaced according to Shandong
32765) obvious inhibitory action is not produced.Therefore, it is very suitable for clinical treatment carrying EGFR L858R according to Shandong for Buddhist nun to dash forward
Change and/or the non-small cell lung cancer of EGFR T790M and/or EGFR delE746_A750 mutation.Further, due to controlling
During treatment to chemotherapeutics produce drug resistance the reason for often with foregoing EGFR L858R and/or EGFR T790M and/or
EGFR delE746_A750 mutation are related, and such drug resistance patient is applied and replaces Buddhist nun to improve the drug resistance of patient according to Shandong
Situation and the effect for playing treatment non-small cell lung cancer.The present invention is to find and complete based on more than.
On the one hand, the present invention relates to be used to treatment carrying EGFR L858R in preparation according to Shandong for Buddhist nun (PCI-32765) be mutated
And/or the use in the medicine of the Patients with Non-small-cell Lung of EGFR T790M mutation and/or EGFR delE746_A750 mutation
On the way.Specifically, the patient can only carry EGFR T790M mutation, or only carry EGFR L858R mutation, or only carry EGFR
DelE746_A750 is mutated, but preferably carries both mutation of EGFR L858R/T790M simultaneously.
Over the course for the treatment of, the medicine according to circumstances can be combined individually or with one or more other therapeutic agents and made
With.The medicament administration that Buddhist nun is replaced according to Shandong can will be included by least one of injection, oral, suction, rectum and applied dermally
To Patients with Non-small-cell Lung.Other therapeutic agents can be selected from following medicine：Immunodepressant (such as tacrolimus, ring spore
Rhzomorph, rapamycin, methotrexate (MTX), endoxan, imuran, mercaptopurine, mycophenolate or FTY720), glucocorticoid
Class medicine (such as metacortandracin, cortisone acetate, prednisolone, methylprednisolone, dexamethasone, betamethasone, fluoxyprednisolone, hydrogen hydroxyl
Prednisolone, beclomethasone, fludrocortisone acetate, percorten, aldosterone), NSAIDs (such as bigcatkin willow
Hydrochlorate, aryl alkanoic acid, 2- arylpropionic acids, N- aryl-anthranilic acids, former times health class, examine former times class or sulphonanilid), it is abnormal anti-
Answer vaccine, antihistamine, anti-leukotriene medicine, beta-2-agonists, theophylline, anticholinergic agent or other selective kinase inhibitors (for example
MTOR inhibitors, c-Met inhibitor) or her2 antibody-drugs.In addition, other therapeutic agents can also be rapamycin
(Rapamycin), gram azoles for Buddhist nun (Crizotinib), TAM, Raloxifene, Anastrozole, Exemestane, Letrozole,
TrastuzumabTM(Herceptin), GleevecTM(Imatinib), taxolTM(taxol), endoxan, Lovastatin, Mei Nuo
Tetracycline (Minosine), cytarabine, 5 FU 5 fluorouracil (5-FU), methotrexate (MTX) (MTX), taxotereTM(docetaxel),
ZoladexTM(Goserelin), vincristine, vincaleukoblastinum, nocodazole, Teniposide, Etoposide, gemzarTM(Ji Xita
Shore), Epothilones (Epothilone), promise only sheet, camptothecine, daunorubicin (Daunonibicin), dactinomycin D, rice support anthracene
Quinone, amsacrine, Doxorubicin (adriamycin), epirubicin or idarubicin.Or, other therapeutic agents can also be thin
Intracellular cytokine such as G-CSF (granulocyte colony stimulating factor).Or, other therapeutic agents can also be such as, but not limited to, CMF
(endoxan, methotrexate (MTX) and 5 FU 5 fluorouracil), CAF (endoxan, adriamycin and 5 FU 5 fluorouracil), AC are (sub-
Baudrillard mycin and endoxan), FEC (5 FU 5 fluorouracil, epirubicin and endoxan), (sub- Baudrillard is mould by ACT or ATC
Element, endoxan and taxol) or CMFP (endoxan, methotrexate (MTX), 5 FU 5 fluorouracil and metacortandracin).
On the other hand, the invention further relates to a kind of pharmaceutical composition, it is included replaces Buddhist nun (PCI-32765) and pharmacy according to Shandong
Upper acceptable carrier or auxiliary agent.Said composition can also be further comprising one or more other therapeutic agents.It is described herein
Other therapeutic agents it is as defined above.
It yet still another aspect, the present invention relates to carry EGFR L858R mutation and/or EGFR using according to Shandong for Buddhist nun's treatment
The method for the Patients with Non-small-cell Lung that T790M is mutated and/or EGFR delE746_A750 are mutated.Over the course for the treatment of, can be with
By injection, oral, suction, rectum or percutaneously for Buddhist nun it will be administered to Patients with Non-small-cell Lung according to Shandong.Can also be according to circumstances
Replace Buddhist nun individually according to Shandong effective dose or be applied in combination with one or more other therapeutic agents.Mentioned other therapeutic agents
It is as defined above.Over the course for the treatment of, it can also be treated using according to Shandong for the chemotherapy joint radiation of Buddhist nun (PCI-32765)
Method is administered.
Brief description of the drawings
Fig. 1 shows the enzyme activity experimental result that Buddhist nun (PCI-32765) is replaced according to Shandong.
Fig. 2 shows the plates of cells Colony forming experimental result that Buddhist nun (PCI-32765) is replaced according to Shandong.
Fig. 3 shows the cell count experimental result that Buddhist nun (PCI-32765) is replaced according to Shandong.
Fig. 4 is shown according to Shandong for Buddhist nun (PCI-32765) to EGFR in NCI-H1975, NCI-H460, A549 and A431 cell
The result of the influence of upstream and downstream path.
Embodiment
Before the present invention is further described, for a better understanding of the present invention, some terms are illustrated.
Definition
" replacing Buddhist nun (Ibrutinib, also referred to as PCI-32765) according to Shandong " is a kind of bruton's tyrosine kinase (Bruton's
Tyrosine kinase, BTK) inhibitor.As a kind of small molecule BTK inhibitor, half that it can be with BTK activated centres
Cystine residue covalent bond, so as to suppress its activity.Have according to Shandong for Buddhist nun with the structure shown in following formula (I)：
Term " EGF-R ELISA " (Epidermal Growth Factor Receptor, EGFR) is a variety of
The tyrosine kinase receptor expressed in epithelial tumor, it is the target spot of oncotherapy.Terms used herein " EGF
Receptor treatment " refers to a kind of cancer therapy for targeting EGF-R ELISA.
Term " EGFR mutation "：The non-small cell lung cancer (NSCLC) of EGFR genetic mutation is to EGF-R ELISA
(EGFR) tyrosine kinase inhibitor (TKI) is very sensitive.Most common EGFR genetic mutation site include No. 18 (G719X),
No. 19 (being lacked in structure) and No. 21 (L858R and L861Q) extrons.EGFR is mutated the drug resistance reaction with TKI drug therapies
It is closely related.
" the L858R mutation " being related in the application is due to that base occurs at the bit codon of exon 21 the 858th to replace, and is led
Cause leucine (L) corresponding with the site in EGFR albumen to become arginine (R), therefore be named as " L858R mutation ".In addition, also
It was found that some mutation in extron 20 appearance --- T790M is mutated.This mutation is due to the bit codon of extron 20 the 790th
Generation base is replaced, and causes threonine (T) corresponding with the site in EGFR albumen to become methionine (M), therefore be named as
" T790M mutation "." the EGFR delE746_A750 mutation " being related in the application is that exons 19 is mutated, as 2235-
The missing of 2249 bit bases, causes 746-750 continuous 5 amino acid deletions (delE746-A750) in EGFR, therefore name
For EGFR delE746_A750 deletion mutations.
Terms used herein " administration " or " administration " include compound is introduced into subject to realize its predetermined function
With the approach of effect.The example for the method for administration that can be used includes injection (hypodermic injection, intravenous injection, parenteral injection, abdomen
Injection, intrathecal injection in film), oral, suction, rectum and percutaneous etc..It can be applied by the form suitable for various methods of administration
Use pharmaceutical preparation.
Terms used herein " pharmaceutically acceptable " refers to, in the range of rational medical judgment, be suitable for
The tissue of people and other mammals contacts without excessive toxicity, stimulation, allergic reaction etc., and with rational interests/wind
The component that danger ratio matches.
Terms used herein " effective dose " is included with regard to dosage and for the necessary time cycle, effectively reaches desired
As a result amount (for example, it is sufficient to treat disease described herein or illness).The effective dose of the compounds of this invention can be according to example
It is as different such as following factor：Morbid state, age and the body weight and compound of subject is in cell or in subject
Cause the ability of desired response.Dosage regimen can be adjusted to provide optimal therapeutic response.
In embodiments of the present invention, according to the present invention to the subject with non-small cell lung cancer apply replaced according to Shandong
When Buddhist nun is treated, the amount for giving medicine depends on factors, such as specific dosage regimen, disease or implant treatment and its sternly
Principal characteristic, the uniqueness (such as body weight) for needing the subject or host treated, still, according to specific ambient conditions, including
The subject or host of the specific medicine, method of administration, the illness for the treatment of and the treatment that have for example used, application dosage can
Routinely determined by methods known in the art.Generally, for the dosage that adult treatment is used, application dosage typically exists
0.02-5000mg/ days, the e.g., from about scope of 1-1500mg/ days.The required dosage can easily be expressed as one or same
When (or in a short time) that is administered or the divided dose at appropriate interval, such as daily two, three, four doses or more point agent.This
Although art personnel are it is understood that give above-mentioned dosage range, the effective dose for replacing Buddhist nun according to Shandong can be according to patient
Situation and combine doctor diagnosed and suitably adjust.
Terms used herein " drug resistance " refers to biological or cell, and (specifically related to tumour cell and tumour is suffered from herein
Person) for pharmaceutically-active tolerance.For example, after tumour cell produces drug resistance to chemotherapeutics, the Chemotherapy of medicine
Just it is decreased obviously.Drug resistance can be divided into acquisition drug resistance and natural bacterial drug resistance according to its occurrence cause.
" IC50 " used herein is also known as half-inhibition concentration, and it refers to obtain maximum in the analysis for measuring certain effect
The 50% of effect suppresses amount, concentration or the dosage of special inhibitor during (such as to the suppression of Btk kinase activities).
The application of the present invention
In certain embodiments of the present invention, applied according to the present invention and treat non-small cell lung cancer according to Shandong for Buddhist nun, it is special
It is not the non-small cell for carrying EGFR L858R mutation and/or EGFR T790M mutation and/or EGFR delE746_A750 mutation
Lung cancer.Treatment can include single therapy, can also include serial therapy.
In certain embodiments of the present invention, before administration replaces Buddhist nun according to Shandong, patient may have gone through epidermis life
Growth factor receptor body is treated.For example, in some specific embodiments, patient had been subjected to the change of tyrosine kinase inhibitor
Learn treatment.In more specifically embodiment, the therapeutic agent used in treatment with tyrosine kinase inhibitors be Gefitinib or
Erlotinib.In some embodiments, patient received Gefitinib or erlotinib treatment 6 months, 7 months, 8 months, 9
Individual month, 10 months, 11 months, 1 year or several years.
In some embodiments that patient has gone through EGF-R ELISA treatment, patient may be to institute
The therapeutic agent (such as Gefitinib and Erlotinib) of use produces drug resistance.In certain embodiments of the present invention, patient couple
The drug resistance of EGF-R ELISA treatment is relevant with EGFR mutation.More specifically, the drug resistance and EGFR L858R and/or
EGFR T790M and/or EGFR delE746_A750 mutation are relevant.In other words, in some specific embodiments, Huan Zheke
Carry one kind in EGFR L858R, EGFR T790M, EGFR delE746_A750 mutation.In preferred embodiment
In, patient can carry both mutation of L858R and T790M simultaneously.According to the present invention, subject is applied can be with for Buddhist nun according to Shandong
Treatment carries the non-small cell of EGFR L858R mutation and/or EGFR T790M mutation and/or EGFR delE746_A750 mutation
Lung cancer.
In certain embodiments of the present invention, can it is to patient daily, the next day or weekly using doses according to Shandong
For Buddhist nun, and continue 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11
The moon, 1 year or several years.It is understood that what patient was applied can be during treating according to patient's feelings for the dosage of Buddhist nun according to Shandong
Condition and Treatment need and suitably increase or decrease.
In certain embodiments of the present invention, simultaneously or sequentially subject can be applied according to Shandong for Buddhist nun and it is a kind of or
A variety of other therapeutic agents.Or, in certain embodiments of the present invention, a kind of drug regimen can be applied to subject
Thing, said composition is formulated as including replaces Buddhist nun and pharmaceutically acceptable carrier or auxiliary agent, and optional one or more according to Shandong
Other therapeutic agents.
For treat non-small cell lung cancer (particularly carry EGFR L858R mutation and/or EGFR T790M mutation and/or
The non-small cell lung cancer of EGFR delE746_A750 mutation) can be formulated into suitable pharmaceutical preparation according to Shandong for Buddhist nun, so as to
For being administered orally (for example, to be dissolved in the such as aqueous liquid form of solvent or water-free liquid or in solid carrier),
Per rectum administration, parenteral, administration in brain pond, Intraperitoneal medication, local administration are (by pulvis, ointment, lotion, solidifying
Jelly, drops, transdermal patch or transdermal skin patches), oral administration, through bronchus form or be used as mouth sprays or nasal spray etc..
Specifically, it can be formulated into for Buddhist nun according to Shandong, for example, for the solution of oral administration, supensoid agent, tablet, dispersible tablet, ball
Agent, capsule, pulvis, sustained release preparation or elixir etc..In the embodiment of drug administration by injection, it can also be configured to close for Buddhist nun according to Shandong
Suitable injection.
Can daily, weekly, monthly, every other month, quarterly or by any other administration schedule with single dose injection or infusion,
Multiple dose is administered with continuous dosage form.The administration of invention formulation can be intermittent to subject, or in gradually
Enter, continuous, constant or controlled speed.In addition, the number of times of the time of form of administration and daily form of administration can not in one day
Together.In certain embodiments of the present invention, be administered to patient can be at 0.02-5000mg/ days, such as Buddhist nun's preparation according to Shandong
The scope of about 1-1500mg/ days.The required dosage can easily be expressed as it is one or be administered simultaneously (or in the short time
It is interior) or divided dose at appropriate interval, such as daily two, three, four doses or more point agent.
For treat non-small cell lung cancer (particularly carry EGFR L858R mutation and/or EGFR T790M mutation and/or
EGFR delE746_A750 mutation non-small cell lung cancer) include according to Shandong for Buddhist nun pharmaceutical composition can be formulated into it is molten
The formulations such as liquid, supensoid agent, tablet, dispersible tablet, pill, capsule, pulvis, sustained release preparation or elixir.Said composition can contain
0.02-5000mg according to Shandong replace Buddhist nun, but using active component effective dose can according to specifically used dosage regimen, give
Medicine approach is different with the order of severity of treated illness and changes.Technical staff is appreciated that for the effective of each patient
Dosage can become XOR metabolic rate difference according to disease severity, individual inheritance and change.However, generally with about 0.5-1000mg
Daily dosage, optional one day 2-4 times divided dose or when giving the compounds of this invention with sustained release forms obtain desired result.
Plan daily accumulated dose about 1-1000mg, about preferably 2-500mg.
The present invention will be illustrated by embodiment and with reference to accompanying drawing below.It should be understood that the embodiment and accompanying drawing that show only are used
Understand the present invention in help, but be not construed as limiting the invention.
Embodiment
Experiment material：WZ4002 is used in embodiment as control.Purchased for Buddhist nun (PCI-32765) and WZ4002 according to Shandong
From Hao Yuan Chemexpress companies (Shanghai).WZ4002 is first well accepted EGFR T790M by checking
Sensitive micromolecular inhibitor is mutated, WZ4002 optionally can not retroactive inhibition mutant egf R (EGFR L858R, EGFR
L858R/T790M), IC50<20nM.WZ4002 also suppresses other mutant egfs R, such as EGFR E746_A750, EGFR E746_
A750/T790M, and it is very weak to normal EGFR cytosiies, therefore side effect is smaller.WZ4002 acts on non-small cell lung cancer
(NSCLC) EGFR, AKT and ERK1/2 phosphorylation (Zhou W, et al., Novel mutant- are suppressed during cell
selective EGFR kinase inhibitors against EGFR T790M.Nature.2009；462(7276):
1070-4)。
Embodiment 1：Influence of the Buddhist nun (PCI-32765) to growth of cancer cells is replaced according to Shandong
Influence of the Buddhist nun (PCI-32765) to growth of cancer cells is replaced according to Shandong by testing, we further assess and replace Buddhist nun according to Shandong
(PCI-32765) selectivity of cancer cell multiplication is suppressed.We have selected Non-small cell lung carcinoma cell NCI- in the present embodiment
H1975 (expression EGFR L858R/T790M double-mutants gene), human lung adenocarcinoma cell NCI-H3255 (expression EGFR L858R
Mutated genes), mouse EGFR-T790M BaF3 cells (stable expression TEL-EGFR-T790M Activating mutations kinases), people source
Non-small cell lung cancer cell PC-9 (expression EGFR delE746_A750 mutated genes), application on human skin squamous cell carcinoma A431 (expression
Wild type EGFR gene), Non-small cell lung carcinoma cell NCI-H460 (expression Wild type EGFR gene), human lung adenocarcinoma cell
A549 (expression Wild type EGFR gene), Non-small cell lung carcinoma cell NCI-H358 (expression Wild type EGFR gene).It is above-mentioned
Cell is purchased from ATCC (U.S.).By various concentrations (0.000508 μM, 0.00152 μM, 0.00457 μM, 0.0137 in embodiment
μM, 0.0411 μM, 0.123 μM, 0.370 μM, 1.11 μM, 3.33 μM, 10 μM) according to Shandong replace Buddhist nun (PCI-32765) and WZ4002
It is added separately in above-mentioned cell, and is incubated 72 hours, uses Cell(Promega, the U.S.) chemistry self-luminous method
Cell viability detection kit, number of viable cells is detected by being quantitative determined to the ATP in living cells.
As a result it is as shown in table 1：Dashed forward according to Shandong for Buddhist nun (PCI-32765) for carrying EGFR T790M mutation, EGFR L858R
The growth inhibition of the non-small cell lung cancer cells of change, EGFR delE746_A750 mutation and the double mutation of EGFR L858R/T790M
Effect is all it is obvious that their IC50 is respectively less than 1 μM, i.e., its inhibition is suitable with WZ4002.And to expression Wild type EGFR
The cell of (EGFR WT), is acted on growing basic unrestraint, its IC50 is all higher than 10 μM according to Shandong for Buddhist nun (PCI-32765)；And
WZ4002 IC50 is more than 4.The result of embodiment 1 supports prominent for carrying EGFR T790M for Buddhist nun (PCI-32765) according to Shandong
Become, EGFR L858R are mutated, EGFR delE746_A750 mutation and the double mutation non-small cell lung cancers of EGFR L858R/T790M are thin
The selectivity of born of the same parents' treatment.
Table 1.PCI-32765 raji cell assay Raji data
Embodiment 2：The external inhibitory activity (enzyme activity) of Buddhist nun (PCI-32765) is replaced according to Shandong
As described below, determined in vitro in enzyme activity measuring according to Shandong for Buddhist nun (PCI-32765) to EGFR (WT), EGFR/
T790M, EGFR L858R/T790M IC50 values.By EGFR intracellular section (699-1068) regional cloning to insect expression vector
In pAcG2T, insect expression system BaculoGold is utilizedTM Baculovirus Expression System(BD
Pharmingen protein expression) is carried out, and with GST labels.T790M and L858R sites are mutated simultaneously, respectively obtained
The double mutational vectors of EGFR T790M single mutation carrier and T790M/L858R.The carrier built is transfected to SF9 packaging virus,
SF9 expressing proteins are infected with virus.
Take the EGFR (WT), EGFR (T790M), the μ L of EGFR (T790M/L858R) protein kinase 9 (6ng/ μ L) of purifying respectively
4 hours (final concentration of 10 μM of medicine, 3 μM, 1 μ are reacted at room temperature with the medicine WZ4002 and PCI-327651 μ L of three times gradient dilution
M、0.3μM、0.1μM、0.03μM、0.01μM、0.003μM)；
Add 2 μ L ATP and 3 μ L substrates Poly (4:1Glu, Tyr) Peptide (Promega, the U.S.) (distinguish by final concentration
For 10 μM and 0.2 μ g/ μ L), 37 DEG C are reacted 1 hour；
The 5 reacted kinase solutions of μ L are taken, 5 μ L ADP-Glo are addedTM(Promega, the U.S.) reagent reacts at room temperature 40min
To terminate kinase reaction and run out of remaining ATP；
Add 10 μ L kinase assay reagents and ADP is changed into ATP, use luciferase/luciferin reaction detection of coupling
The ATP newly synthesized, maps using after Envision readings, calculates IC50 values.
Experimental result is as shown in Figure 1：Have according to Shandong for Buddhist nun (PCI-32765) to EGFR L858R/T790M double-mutants albumen
There are strong inhibitory action, IC50=3nM；There is obvious inhibitory action, IC50=to EGFR T790M single mutation type albumen
50.77nM.Its exercising result is suitable with WZ4002.These results explanation is T790M saltant types for Buddhist nun (PCI-32765) according to Shandong
EGFR and L858R/T790M double-mutants EGFR inhibitor.
Embodiment 3：Plate clone formation experiment
Using conventional pancreatin had digestive transfer culture method, by the double mutation of the carrying EGFR L858R/T790M of exponential phase of growth
Cell suspending liquid is made in NCI-H1975 Non-small cell lung carcinoma cells.Cell suspending liquid is blown and beaten repeatedly, makes cell fully dispersed.
To carrying the NCI-H1975 Non-small cell lung carcinoma cell counts of the double mutation of EGFR L858R/T790M, and adjusted with culture medium
Cell concentration.
According to the propagation energy for the NCI-H1975 Non-small cell lung carcinoma cells for carrying the double mutation of EGFR L858R/T790M
Power, by 105The concentration of cells/well is inoculated into six orifice plates (diameter 4cm) of the culture medium containing 2mL, is gently rocked with ten word directions
Culture dish, makes cell be uniformly dispersed.
Culture dish is placed in 37 DEG C, 5%CO2Middle culture is administered after 24 hours, and drug concentration is as follows：Buddhist nun (PCI- is replaced according to Shandong
32765) it is 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM in DMSO；Blank control uses the DMSO of same volume.
After culture 72 hours, culture is terminated, nutrient solution is discarded, PBS liquid carefully embathes 2 times.15 minutes are fixed with methanol, is abandoned
Remove methanol.Use violet staining.
Experimental result is as shown in Figure 2：Just substantially suppress cell collection at 0.1 μM with WZ4002 according to Shandong for Buddhist nun (PCI32765)
The formation fallen, and can consumingly suppress the formation of cell colony at 1 μM, the substantially acellular colony shape at 10 μM
Into.
Embodiment 4：Cell count is tested
Using conventional pancreatin had digestive transfer culture method, by the double mutation of the carrying EGFR L858R/T790M of exponential phase of growth
Cell suspension is made in NCI-H1975 non-small cell lung cancer cells.Cell suspension is blown and beaten repeatedly, makes cell fully dispersed.To cell
Numeration, and adjust cell concentration with culture medium.
According to the multiplication capacity for carrying the NCI-H1975 non-small cell lung cancer cells that EGFR L858R/T790M are mutated, press
105The concentration of cells/well is inoculated into six orifice plates (diameter 4cm) of the culture medium containing 2mL, and culture is gently rocked with ten word directions
Ware, makes cell be uniformly dispersed.
Culture dish is placed in 37 DEG C, 5%CO2Middle culture is administered after 24 hours, and drug concentration is as follows：Buddhist nun (PCI- is replaced according to Shandong
32765) it is 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM in DMSO；Blank control uses the DMSO of same volume.
After culture 72 hours, culture is terminated, nutrient solution is discarded, PBS liquid carefully embathes 2 times, added after pancreatin digestion to thin
Born of the same parents are counted, and IC50 values are calculated according to cell quantity.
Experimental result is as shown in Figure 3：Be computed, according to Shandong for Buddhist nun (PCI-32765) IC50 values for 172.7nM, illustrate according to
Shandong is played a part of suitable with WZ4002 for Buddhist nun (PCI-32765), is all shown to non-small cell lung cancer cell NCI-H1975
The growth inhibition effect of (EGFR L858R/T790M).
Embodiment 5：Influence of the Buddhist nun (PCI-32765) to upstream and downstream signal path in cell is replaced according to Shandong
By determining many cellular biochemistry terminals and feature terminal, we have evaluated replaces Buddhist nun (PCI- according to Shandong
32765) influence pair relative with suppressing EGFR closely related protein kinase AKT, ErK, STAT3.With 0.03 μM of various concentrations,
0.1 μM, 0.3 μM, 1 μM, 3 μM handled respectively for Buddhist nun (PCI-32765) according to Shandong carries the double mutation of EGFR L858R/T790M
NCI-H1975 Non-small cell lung carcinomas cell (EGFR L858R/T790M), application on human skin squamous cell carcinoma A431 (EGFR WT), people
Non-small cell lung cancer cell NCI-H460 (EGFR WT), human A549 cell lines (EGFR WT) 4 hours, collect sample.Survey
Determine influence of the compound to the signal path of this four cell lines.
Experimental result is as shown in Figure 4：Carrying NCI-H1975 people's non-small cell lung of the double mutation of EGFR L858R/T790M
In cancer cell, according to Shandong for Buddhist nun (PCI-32765) and WZ4002 can effectively suppress EGFR-Tyr1068, AKT-Ser473,
AKT-Thr308, ERK-Thr202/Tyr204 phosphorylation, inhibiting rate is more than 50% when replacing Buddhist nun's concentration according to Shandong for 100nM.And
(application on human skin squamous cell carcinoma A431 (EGFR WT), Non-small cell lung carcinoma are thin in three cell lines of expression Wild type EGFR gene
Born of the same parents NCI-H460 (EGFR WT), human A549 cell lines (EGFR WT), according to Shandong for Buddhist nun and WZ4002 all to corresponding signal
Path is substantially without influence.These results illustrate to replace the inhibitor that Buddhist nun and WZ4002 are mutant egf R according to Shandong.Also enter one simultaneously
Step is demonstrated according to Shandong for Buddhist nun (PCI-32765) to carrying the double NCI-H1975 non-small cell lungs being mutated of EGFR L858R/T790M
The growth inhibition effect and selectivity of cancer cell.
Claims (9)
1. prepared according to Shandong for Buddhist nun for treating carrying EGFR T790M mutation and/or EGFR L858R mutation and/or EGFR
Purposes in the medicine of the Patients with Non-small-cell Lung of delE746_A750 mutation.
2. purposes according to claim 1, wherein the Patients with Non-small-cell Lung carries EGFR L858R and EGFR
The double mutation of T790M.
3. purposes according to claim 1 or 2, wherein being administered alone or being controlled with one or more others for Buddhist nun according to Shandong
Agent is treated to be administered in combination.
4. a kind of suppression of non-treatment purpose carries EGFR L858R mutation and/or EGFR T790M mutation and/or EGFR
The method of the non-small cell lung cancer cell of delE746_A750 mutation, including the cell is in contact with according to Shandong for Buddhist nun.
5. method as claimed in claim 4, wherein the cell is selected from Non-small cell lung carcinoma cell NCI-H1975, people's lung
Adenocarcinoma cell NCI-H3255, mouse EGFR-T790M BaF3 cells and people source non-small cell lung cancer cell PC-9.
6. method as claimed in claim 4, wherein the cell carries EGFR L858R mutation and/or EGFR T790M are prominent
Become.
7. prepared according to Shandong for Buddhist nun for suppressing to carry EGFR L858R mutation and/or EGFR T790M mutation and/or EGFR
Purposes in the medicine of the non-small cell lung cancer cell of delE746_A750 mutation.
8. purposes as claimed in claim 7, wherein the cell is selected from Non-small cell lung carcinoma cell NCI-H1975, people's lung
Adenocarcinoma cell NCI-H3255, mouse EGFR-T790M BaF3 cells and people source non-small cell lung cancer cell PC-9.
9. purposes as claimed in claim 7, wherein the cell carries the double mutation of EGFR L858R and EGFR T790M.
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| CN201410258423.3A CN104825455B (en) | 2014-06-11 | 2014-06-11 | The purposes of Buddhist nun is replaced according to Shandong |
| PCT/CN2015/081050 WO2015188738A1 (en) | 2014-06-11 | 2015-06-09 | New use of ibrutinib |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102482277A (en) * | 2009-05-05 | 2012-05-30 | 达纳-法伯癌症研究所有限公司 | Egfr inhibitors and methods of treating disorders |
| WO2013059738A2 (en) * | 2011-10-19 | 2013-04-25 | Pharmacyclics, Inc. | Use of inhibitors of bruton's tyrosine kinase (btk) |
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| CA2829669A1 (en) * | 2013-10-07 | 2015-04-07 | Miami Mice Research Corp | Btk and hdac inhibitors to treat non-hematologic cancers |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102482277A (en) * | 2009-05-05 | 2012-05-30 | 达纳-法伯癌症研究所有限公司 | Egfr inhibitors and methods of treating disorders |
| WO2013059738A2 (en) * | 2011-10-19 | 2013-04-25 | Pharmacyclics, Inc. | Use of inhibitors of bruton's tyrosine kinase (btk) |
Non-Patent Citations (2)
| Title |
|---|
| In Vitro and In Vivo Characterization of Irreversible Mutant-Selective EGFR Inhibitors That Are Wild-Type Sparing;Sjin, RTT, et al.;《MOLECULAR CANCER THERAPEUTICS》;20140430(第13期);摘要 * |
| Novel mutant-selective EGFR kinase inhibitors against EGFR T790M;Wenjun Zhou, et al.;《NATURE》;20091231;第462卷;1070-1074 * |
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