CN104873515B - The application of acute leukemia FLT3 ITD being mutated for Buddhist nun according to Shandong - Google Patents
The application of acute leukemia FLT3 ITD being mutated for Buddhist nun according to Shandong Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—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
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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
- A61K31/52—Purines, e.g. adenine
Abstract
The invention discloses specifically, present invention discover that according to Shandong for Buddhist nun can be used for treat acute myelocytic leukemia, the acute myelocytic leukemia of FLT3/ITD mutated genes is particularly carried for a kind of new application of Buddhist nun (PCI32765) according to Shandong.
Description
Technical field
The present invention relates to field of medicaments, more particularly to replaces a kind of new application of Buddhist nun (PCI32765) according to Shandong.
Background technology
Acute myelocytic leukemia (acute myelocytic leukemia, AML) or acute non lymphocytic leukemia
(ANLL) including the acute leukemia in all non-lymphocytes source.It is multipotential stem cell or the precursor for slightly having broken up
Caryogram is undergone mutation formed a class disease, is the Clonal malignant disease of hemopoietic system.Epidemiology survey shows, ring
The onset relation of border, occupation and inherent cause and AML is close.The incidence of disease of developed country is higher than developing country, western countries
Higher than oriental countries.Annual morbidity is 2.25/10 ten thousand populations all over the world, and with age increase, the incidence of disease increases, and 50 years old starts
Substantially rise, reach within 60～69 years old peak, be within less than 30 years old 1/,100,000, be then up to 17/,100,000 within more than 75 years old.Therefore, AML is really
In one kind, disease of old people, account for the 80%～90% of adult acute leukemia, but only account for the 15%～20% of acute leukemia.
Meanwhile, male sex's morbidity is higher than women.
FLT3 (Fms-like tyrosine kinase 3) i.e. FMS samples EGFR-TKs 3, with c-Kit, c-FMS and
PDGFR belongs to type III receptor tyrosine kinase (receptor tyrosine kinase III, RTK III) family member,
Its protein structure includes guarantor's outskirt of 5 immunoglobulin (Ig) (Ig) spline structure domains composition, 1 transmembrane region, 1 membrane-proximal region (JM),
And 2 EGFR-TK (TK) areas that intracellular is separated by kinase insert (S.D.Lyman etc., Oncogene, 1993,
8,815-822).It is found that within 1996 that FLT3 is mutated first in AML cells, its mutation type is that internal series-connection repeats (FLT3/
ITD).In recent years, many researchs have confirmed that the activated mutant of FLT3 plays very heavy in the generation of AML and the progress of disease
The pathological effect wanted.AML patient with FLT3/ITD activated mutants generally has leukocyte counts high, clinical prognosis
It is poor, easy unique Clinical symptoms such as recurrence, and due to the detection method of FLT3 activated mutants it is simple, it is therefore more and more
Researcher be devoted to for FLT3 developing into the conventional detection means of AML for instructing sentencing for the treatment and prognosis of AML patient
Break and as the detection means of parvovirus B19, and as the another new target of leukaemic's chemotherapeutics
Point.
Confirm that the activated mutant of FLT3 mainly there are two kinds：Internal series-connection repeats (internal tandem
Duplication, ITD) and activate point mutation (point mutation in the activation loop, the Tkd in ring
Point mutation).Both activated mutants of FLT3 are responsible for FLT3 be there is autophosphorylation and then causes FLT3 generation parts non-
Dependent constitutively activated, further activates signal transduction abnormal downstream, promotes propagation and suppression apoptosis so as to play
Effect so that the leukaemic's clinical prognosis with this mutant phenotype are poor.
Study hotspot is become to the targeted inhibition of FLT3 gene mutations at present, small molecule tyrosine kinase suppression is predominantly developed
Preparation, suppresses its activity by competing ATP-binding site with FLT3 EGFR-TKs.The suppression of clinic is come at present
The kinase inhibitor of FLT3 has AC220 etc..
It is a kind of bruton's tyrosine kinase (Bruton's for Buddhist nun (Ibrutinib, also referred to as PCI32765) according to Shandong
Tyrosine kinase, BTK) inhibitor, which can be used to be used alone or in the way of being used with other therapeutic agents
In treatment autoimmune disease or illness, heteroimmunity disease or illness, cancer include lymthoma and inflammatory disease or
Illness.At present, yet there are no and for Buddhist nun (PCI32765) treat the acute marrow that carry FLT3/ITD mutated genes according to Shandong with regard to using
The relevant report of chronic myeloid leukemia.
The content of the invention
In view of aforesaid technical problem, inventor conducts extensive research.As a result, inventor is it was unexpectedly observed that according to Shandong
Can effectively treat for this bruton's tyrosine kinase inhibitor of Buddhist nun (PCI32765) and carry FLT3/ITD mutated genes
Acute myelocytic leukemia.More specifically, inventor has found, the acute marrow according to Shandong for Buddhist nun to carrying FLT3/ITD mutated genes
Cell leukemia cell such as MOLM-13, MOLM-14, MV-4-11 play stronger inhibitory action (IC50 be respectively 0.47 μM,
0.66 μM, 0.33 μM), and it is thin to the acute marrow for carrying FLT3WT (wild type) and FLT3 A680V mutated genes for Buddhist nun according to Shandong
The all no obvious inhibitory action of born of the same parents leukaemia.Therefore clinical treatment is applicable to for Buddhist nun according to Shandong and carries FLT3/ITD mutation
The acute myelocytic leukemia of type gene.
On the one hand, the present invention relates to prepare for treating carrying FLT3/ITD saltant type bases for Buddhist nun (PCI32765) according to Shandong
Purposes in the medicine of the acute myelocytic leukemia cancer patient of cause.
Over the course for the treatment of, the medicine according to circumstances can be made individually or with one or more other therapeutic combination
With.Can pass through at least one by comprising the medicament administration that Buddhist nun is replaced according to Shandong in injection, oral, suction, rectum and applied dermally
To the acute myelocytic leukemia patient for carrying FLT3/ITD mutators.Other therapeutic agents can be selected from following medicine：Exempt from
Epidemic disease inhibitor (such as tacrolimus, encircles rhzomorph, rapamycin, Methylaminopterin, endoxan, imuran, mercaptopurine, wheat
Examine phenolic ester or FTY720), glucocorticoids medicine (for example metacortandracin, cortisone acetate, prednisolone, methylprednisolone, fill in rice
Pine, betamethasone, fluoxyprednisolone, hydrogen hydroxyl prednisolone, beclomethasone, fludrocortisone acetate, percorten, aldehyde are solid
Ketone), NSAIDs (for example salicylate, aryl alkanoic acid, 2- arylpropionic acids, N- aryl-anthranilic acids, former times health class,
Examine former times class or sulphonanilid), allergic reaction bacterin, antihistamine, anti-leukotriene medicine, beta-2-agonists, theophylline, anticholinergic agent or
Other selective kinase inhibitors (such as mTOR inhibitors, c-Met inhibitor) or her2 antibody-drugs.In addition, mentioned
Other therapeutic agents can also be rapamycin (Rapamycin), gram azoles for Buddhist nun (Crizotinib), TAM, Raloxifene,
Anastrozole, Exemestane, Letrozole, TrastuzumabTM(Herceptin), GleevecTM(Imatinib), taxolTM(Japanese yew
Alcohol), endoxan, Lovastatin, U.S. promise tetracycline (Minosine), cytarabine, 5 FU 5 fluorouracil (5-FU), methotrexate (MTX)
(MTX), taxotereTM(docetaxel), ZoladexTM(Goserelin), vincristine, vincaleukoblastinum, nocodazole, for Ni Bo
Glycosides, Etoposide, gemzarTM(gemcitabine), Epothilones (Epothilone), promise only sheet, camptothecine, daunorubicin
(Daunonibicin), dactinomycin D, mitoxantrone, amsacrine, Doxorubicin (adriamycin), epirubicin or she reach
Compare star.Or, other therapeutic agents can also be cell factor such as G-CSF (granulocyte colony stimulating factor).Or, other are controlled
Treating agent can also be, such as but not limited to, CMF (endoxan, methotrexate (MTX) and 5 FU 5 fluorouracil), CAF (endoxan, Asia
Baudrillard mycin and 5 FU 5 fluorouracil), AC (adriamycin and endoxan), FEC (5 FU 5 fluorouracil, epirubicin and
Endoxan), ACT or ATC (adriamycin, endoxan and taxol) or CMFP (endoxan, methotrexate (MTX), 5-
Fluorouracil and metacortandracin).
On the other hand, the invention further relates to a kind of pharmaceutical composition, which is included according to Shandong for Buddhist nun (PCI32765) and pharmacy
Upper acceptable carrier or auxiliary agent.Said composition can also further include one or more other therapeutic agent.It is described herein
Other therapeutic agents it is as defined above.
It yet still another aspect, the present invention relates to using thin for the acute marrow that Buddhist nun treats carrying FLT3/ITD mutated genes according to Shandong
The method of born of the same parents leukaemic.Over the course for the treatment of, percutaneously will can apply by injection, oral, suction, rectum or for Buddhist nun according to Shandong
With to the acute myelocytic leukemia patient for carrying FLT3/ITD mutated genes.Can also be according to circumstances by effective dose according to Shandong
Use individually or with one or more other therapeutic combination for Buddhist nun.Mentioned other therapeutic agents are as defined above.
Over the course for the treatment of, the chemotherapy joint radiotherapy according to Shandong for Buddhist nun (PCI32765) can also will be used to be administered.
It yet still another aspect, a kind of the present invention relates to acute myelocytic leukemia for suppressing to carry FLT3/ITD mutated genes
The method of cell, including by the cell with contact for Buddhist nun according to Shandong.According to the present invention, FLT3/ITD mutated genes are carried
Acute Myeloid Leukemia Cells Contributing is preferably selected from the one kind in MOLM-13, MOLM-14, MV-4-11, PL-21 and MUTZ-11
Or it is various.It is further preferred that the cell and valid density are at least 0.05 μM contact for Buddhist nun according to Shandong, more preferably according to
Shandong is at least 0.1 μM, further preferred 0.3-10 μM, such as 0.3-3 μM for the amount of Buddhist nun.
Description of the drawings
Fig. 1 illustrates PCI32765 respectively on MOLM-14 (1a) and OCI-AML-3 (1b) cell to BTK signal paths
Affect.
Fig. 2 illustrates that PCI32765 is right on MOLM-13 (2a), MOLM-14 (2b) and MV-4-11 (2c) cell respectively
The impact of FLT3/ITD relatively closely related albumen and associated signal paths.
Fig. 3 illustrates closely related albumen relative with FLT3/ITD in cell.
Fig. 4 illustrates PCI32765 respectively to MOLM-13 (4a), MOLM-14 (4b), MV-4-11 (4c), OCI-AML-3
(4d) the apoptotic impact with NOMO-1 (4e).
Fig. 5 illustrates PCI32765 respectively to MOLM-14 (5a), MV-4-11 (5b), MOLM-13 (5c), OCI-AML-3
(5d) impact with the cell cycle distribution of NOMO-1 (5e).
Specific 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 PCI32765) according to Shandong " is a kind of bruton's tyrosine kinase (Bruton's
Tyrosine kinase, BTK) inhibitor.Used as a kind of small molecule BTK inhibitor, it can be with the half of BTK activated centres
Cystine residue covalent bond, so as to suppress its activity.According to Shandong for Buddhist nun with the structure shown in following formula (I)：
FLT3 (Fms-like tyrosine kinase 3) i.e. FMS samples EGFR-TKs 3.FLT3/ITD mutators or
FLT3/ITD mutated genes refer to transmembrane region internal series-connection repeat (FLT3 internal tandem duplication,
FLT3/ITD) be mutated, be in acute myeloid leukemia (acute myeloid leukaemia, AML) incidence highest and with it is pre-
Related mutation afterwards.
Terms used herein " administration " or " administration " include compound is introduced in experimenter to realize its predetermined function
With the approach of effect.The example of the method for administration that can be used includes injecting (hypodermic injection, intravenous injection, parenteral injection, abdomen
Injection, intrathecal injection in film), oral, suction, rectum and percutaneous etc..Can be applied by being suitable to the form of various methods of administration
Use pharmaceutical preparation.
Terms used herein " pharmaceutically acceptable " is referred to, in the range of rational medical judgment, be suitable for
The contact tissue of people and other mammals without excessive toxicity, stimulation, allergic reaction etc., and with rational interests/wind
The component that danger ratio matches.
Terms used herein " effective dose " includes with regard to dosage and for the necessary time cycle, effectively reaches desired
As a result the amount of (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：The morbid state of experimenter, age and body weight and compound are in cell or in experimenter
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 carry FLT3/ITD mutated genes acute marrow
The experimenter of chronic myeloid leukemia is applied when being treated for Buddhist nun according to Shandong, and the amount for giving medicine depends on factors, such as specific
The uniqueness (such as body weight) of dosage regimen, disease or implant treatment and its seriousness, subject in need for the treatment of or host,
But, according to specific ambient conditions, including the concrete medicine, method of administration, the illness for the treatment of and treatment that have for example adopted
Subject or host, application dosage routinely can be determined by methods known in the art.Generally, the agent for adult treatment being used
For amount, application dosage typically at 0.02-5000mg/ days, the e.g., from about scope of 1-1500mg/ days.The required dosage can be with
One, or (or at short notice) that be administered simultaneously or in the divided dose at appropriate interval is expressed as easily, for example daily
2nd, three, four doses or more points of agent.Although it will be appreciated by persons skilled in the art that give above-mentioned dosage range, according to Shandong
Effective dose for Buddhist nun can be suitably adjusted according to the situation of patient and with reference to doctor diagnosed.
" IC50 " used herein, also known as half-inhibition concentration, which refers to and obtains maximum in the analysis for measuring certain effect
The amount of special inhibitor, concentration or dosage during 50% suppression (such as the suppression to BTK kinase activities) of effect.
" EC50 " used herein refers to dosage, concentration or the amount for determining compound, and which causes particular assay compound to lure
The dose-dependant reaction of the maximum expression of the 50% of the specific reaction led, stimulate or strengthen.
The application of the present invention
In certain embodiments of the present invention, applied according to the present invention FLT3/ITD mutation is carried to treat for Buddhist nun according to Shandong
The acute myelocytic leukemia of type gene.Treatment can include single therapy, it is also possible to including serial therapy.
In certain embodiments of the present invention, can it is daily to patient, the next day or weekly apply 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 experimenter can be applied according to Shandong for Buddhist nun and it is a kind of or
Various other therapeutic agents.Or, in certain embodiments of the present invention, a kind of drug regimen can be applied to experimenter
Thing, said composition are formulated as replacing Buddhist nun and pharmaceutically acceptable carrier or auxiliary agent, and optional one or more comprising according to Shandong
Other therapeutic agents.
For treating being formulated for Buddhist nun according to Shandong for the acute myelocytic leukemia for carrying FLT3/ITD mutated genes
It is into suitable pharmaceutical preparation, (for example, such as aqueous or water-free to be dissolved in the liquid form of solvent for use in being administered orally
Liquid or in solid carrier), per rectum administration, parenteral, administration in brain pond, Intraperitoneal medication, local administration it is (logical
Cross pulvis, ointment, lotion, gel, drops, transdermal patch or transdermal skin patches), oral administration, Jing bronchuses form or work
For mouth sprays or nasal spray etc..Specifically, can be formulated into for Buddhist nun according to Shandong, for example, for the solution of oral administration, mix
Suspension, tablet, dispersible tablet, pill, capsule, pulvis, sustained release preparation or elixir etc..In the embodiment of drug administration by injection, according to
Shandong can also be configured to suitable injection for Buddhist nun.
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 experimenter, or in gradually
Enter, continuous, constant or controlled speed.Additionally, 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 easily can be expressed as it is one, or be administered simultaneously (or in the short time
It is interior) or in the divided dose at appropriate interval, such as daily two, three, four doses or more points of agent.
For treat carry FLT3/ITD mutated genes acute myelocytic leukemia comprising according to Shandong for Buddhist nun medicine
Composition can be formulated into the agent such as solution, supensoid agent, tablet, dispersible tablet, pill, capsule, pulvis, sustained release preparation or elixir
Type.Said composition can replace Buddhist nun according to Shandong containing 0.02-5000mg, but the effective dose of used active component can be according to tool
Dosage regimen that body is used, method of administration are different with the order of severity of treated illness and change.Technical staff can manage
Solution, the effective dose for each patient can become XOR metabolic rate difference according to disease severity, individual inheritance and change.So
And, generally with the daily dosage of about 0.5-1000mg, optional one day 2-4 time divided dose or chemical combination of the present invention is given with sustained release forms
During thing, desired result is obtained.Plan daily accumulated dose about 1-1000mg, preferably about 2-500mg.
By embodiment and accompanying drawing will be combined to illustrate the present invention below.It should be understood that the embodiment for illustrating and accompanying drawing are only used
The present invention is understood in help, but be not construed as limiting the invention.
Embodiment
Experiment material：Used in embodiment, AVL-292, CGI1746 and AC220 are used as control.AVL-292 is by altogether
The BTK inhibitor with high selectivity that valency is combined, its IC50 are less than 0.5nM (Robak T, Robak E, " Tyrosine
kinase inhibitors as potential drugs for B-cell lymphoid malignancies and
autoimmune disorders”,Expert.Opin.Investig.Drugs,2012,21(7):921-947)。CGI1746
The small molecule BTK kinase inhibitor of new high selectivity, its IC50 be 1.9nM (Di Paolo JA, et al.,
“Specific Btk inhibition suppresses B cell-and myeloid cell-mediated
arthritis”,Nat.Chem.Biol.,2011,7(1):41-50).AC220 is the FLT3 kinases for coming into clinic at present
Inhibitor.Buddhist nun (PCI32765) and AVL292, CGI1746 and AC220 is replaced to be purchased from Hao Yuan Chemexpress according to Shandong public
Department (Shanghai).
Embodiment 1：Impact of the Buddhist nun (PCI32765) to cancer cell multiplication is replaced according to Shandong
Impact of the Buddhist nun (PCI32765) to growth of cancer cells is replaced according to Shandong by test, is further assessed and Buddhist nun is replaced according to Shandong
(PCI32765) selectivity of inhibition cancer cell propagation.Acute myeloid leukemia cells in children MOLM-16 (tables have been selected in the present embodiment
Up to wild type FLT3 gene), acute myeloid leukemia cells in children HL-60 (expression wild type FLT3 gene), acute myeloid leukaemia
Cell KASUMI-1 (expression wild type FLT3 gene), acute promyelocytic leukemia cell strain NB-4 (Lu+) (are expressed wild
Type FLT3 gene), human muscle creatine kinase cell line OCI-AML-2 (expression FLT3 A680V mutated genes), people it is acute
Marrow leukaemia cell strain OCI-AML-3 (expression FLT3 A680V mutated genes), human muscle creatine kinase cell line
MOLM-14 (expression FLT3/ITD mutated genes and wild type FLT3 gene), human muscle creatine kinase cell line MOLM-13
(expression FLT3/ITD mutated genes and wild type FLT3 gene), people acute monocytic leukemia cell line MV-4-11
(expression FLT3/ITD mutated genes), human muscle creatine kinase cell line NOMO-1 (expression wild type FLT3 gene),
MDS-RAEB (myelodysplastic syndrome-initial cell increases type) cell line SKM-1 (expression wild type FLT3 gene), people
Acute myeloid leukemia cells in children strain U-937 (expression wild type FLT3 gene), the acute red white corpuscle leukemia cell line HEL of people
(expression wild type FLT3 gene), the acute macronucleus chronic myeloid leukemia CMK of people (expression wild type FLT3 gene), people's Epstein-Barr virus sense
The human peripheral lymphocyte JVM-2 of dye, people Burkitt's lymphoma cell Namalwa, people lymphoma mantle cell (mantel
Cell lymphoma, MCL) cell REC-1, people's lymphoma mantle cell (mantel cell lymphoma, MCL) cell Z-
138th, people's diffusivity tissue lymph oncocyte SU-DHL-2, people's diffusivity large B cell lymphoid tumor clone TMD8, human B cell are slow
Property lymphoblastic leukemia cell lines MEC-1, human B cell chronic lymphocytic leukemia cell line MEC-2, people's B- cellularitys are white
Blood disease cell line NALM-6, people Pancytopenia cell line JURKAT and blood cell strain K562.Above cell is equal
Purchased from ATCC.
By variable concentrations (0.000508 μM, 0.00152 μM, 0.00457 μM, 0.0137 μM, 0.0411 μ in embodiment
M, 0.123 μM, 0.370 μM, 1.11 μM, 3.33 μM, 10 μM in DMSO) be added to for Buddhist nun (PCI32765) according to Shandong it is above-mentioned thin
In born of the same parents, and it is incubated 72 hours, uses Cell Titer-The cell viability detection examination of (Promega, the U.S.) chemistry self-luminous method
Agent box, detects number of viable cells by carrying out quantitative determination to the ATP in living cells.
Experimental result as shown in table 1, is found according to Shandong for Buddhist nun (PCI32765) only to expressing FLT3/ITD mutated genes
Human muscle creatine kinase cell line MOLM-14 (expression FLT3/ITD mutated genes and wild type FLT3 gene), people are acute
Marrow leukaemia cell strain MOLM-13 (expression FLT3/ITD mutated genes and wild type FLT3 gene), the acute monokaryon of people are thin
The propagation of born of the same parents' property leukemia cell line MV-4-11 (expression FLT3/ITD mutated genes) this three plants of cancer cells is with strong suppression
Make and use, 0.47 μ is respectively to the half-inhibition concentration IC50 of cell line MOLM-13, MOLM-14 and MV-4-11 for Buddhist nun according to Shandong
M, 0.66 μM, 0.33 μM, and (half-inhibition concentration IC50 values are all higher than 2 μ to the equal unrestraint effect of the propagation of other cancer cells
M).The result of embodiment 1 supports thin for the acute marrow for carrying FLT3/ITD mutated genes for Buddhist nun (PCI32765) according to Shandong
The selectivity of the white blood treatment of born of the same parents.
Determination datas of the table 1.PCI32765 on cancer cell
| Clone | Cell type | FLT3 types | PCI32765IC50(μM) |
| MOLM-16 | AML-M0 | FLT3WT | 4.4 |
| HL-60 | AML-M2 | FLT3WT | 2.1 |
| KASUMI-1 | AML-M2 | FLT3WT | 3.0 |
| NB-4 | AML-M3 | FLT3WT | 7.5 |
| OCI-AML-2 | AML-M4 | FLT3 A680V | >10 |
| OCI-AML-3 | AML-M4 | FLT3 A680V | >10 |
| MOLM-13 | AML-M5a | FLT3/ITD,FLT3WT | 0.47 |
| MOLM-14 | AML-M5a | FLT3/ITD,FLT3WT | 0.66 |
| MV-4-11 | AML-M5 | FLT3/ITD | 0.33 |
| NOMO-1 | AML-M5 | FLT3WT | >10 |
| SKM-1 | AML-M5 | FLT3WT | 8.6 |
| U-937 | AML-M5 | FLT3WT | 8.5 |
| HEL | AML-M6 | FLT3WT | >10 |
| CMK | AML-M7 | FLT3WT | >10 |
| JVM-2 | MCL | -- | >10 |
| Namalwa | Burkitt lymphoma | -- | >10 |
| REC-1 | MCL | -- | >10 |
| Z-138 | MCL | -- | >10 |
| SU-DHL-2 | ABC DLBCL | -- | >10 |
| TMD8 | ABC DLBCL | -- | >10 |
| MEC-1 | B-CLL | -- | >10 |
| MEC-2 | B-CLL | -- | >10 |
| NALM-6 | ALL | -- | >10 |
| JURKAT | ALL | -- | >10 |
| K562 | CML | -- | >10 |
Embodiment 2：Impact of the Buddhist nun (PCI32765) in cell to BTK upstream and downstream signal paths is replaced according to Shandong
Acute myeloid leukemia cells in children MOLM-14 (expression FLT3/ITD mutated genes and wild type FLT3 gene) with
And on acute myeloid leukemia cells in children OCI-AML-3 (expression FLT3 A680V mutated genes) this two plants of cells, by determining
Many cellular biochemistry terminals and feature terminal, have evaluated according to Shandong for Buddhist nun (PCI32765) to BTK kinases in cell and
The impact of protein kinase PLC γ, AKT, ErK, GSK3 β closely related with BTK kinases.With 0 μM of variable concentrations, 0.3125 μM,
0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM (in DMSO) according to Shandong replace Buddhist nun (PCI32765), 1 μM (in DMSO's)
AVL-292, and the CGI1746 of 1 μM (in DMSO) process respectively acute myeloid leukemia cells in children MOLM-14 (expression FLT3/
ITD mutated genes and wild type FLT3 gene), acute myeloid leukemia cells in children OCI-AML-3 (expression FLT3 A680V mutation
Type gene) 4 hours, and employment Immunoglobulin IgM stimulates 10min, collects sample.Compound is determined in the two clones
The impact (Fig. 1) of 42/44 phosphorylation of BTKY223, PLC γ Y1217, AKT308, AKT S473, Erk.
Experimental result is as shown in Figure 1：No matter in acute myeloid leukemia cells in children MOLM-14 (expression FLT3/ITD saltant types
Gene and wild type FLT3 gene) in, or in acute myeloid leukemia cells in children OCI-AML-3 (expression FLT3 A680V mutation
Type gene) in cell, PLC γ of the Buddhist nun (PCI32765) although the phosphorylation of BTK can be suppressed, to BTK downstreams is replaced according to Shandong
Phosphorylation have no effect (Fig. 1), and the phosphorylation of AKT, Erk is not also affected.This explanation replaces Buddhist nun (PCI32765) according to Shandong
Affect to carry the acute myeloid leukaemia of FLT3/ITD mutated genes not by the phosphorylation of suppression protein kinase B TK
The cell propagation of cell line MOLM-14.
Embodiment 3：Impact of the Buddhist nun (PCI32765) in cell to FLT3 upstream and downstream signal paths is replaced according to Shandong
Carry FLT3/ITD mutated genes acute myeloid leukemia cells in children MOLM-13, MOLM-14, MV-4-11 this
In three plants of cells, by determining many cellular biochemistry terminals and feature terminal, test and Buddhist nun (PCI32765) is replaced according to Shandong
Phosphorylation and its closely related signal path downstream STAT5 albumen to the protein kinase of the FLT3/ITD saltant types in cell
The impact (Fig. 3) of phosphorylation, other related Protein kinase ERKs, the impact of AKT phosphorylations, while we are also have detected to egg
The impact of white C-Myc and transcription factor NF-KB subunit p65 and its phosphorylation.With 0 μM of variable concentrations, 0.03 μM, 0.1 μM,
The FLT3 kinase inhibitor AC220 that Buddhist nun (PCI32765), 0.1 μM (in DMSO) are replaced according to Shandong of 0.3 μM, 1 μM (in DMSO),
The BTK kinase inhibitor AVL-292 of 1 μM (in DMSO), the BTK kinase inhibitor CGI1746 of 1 μM (in DMSO) locate respectively
The acute myeloid leukemia cells in children of tri- plants of carrying FLT3/ITD mutated genes of reason MOLM-13, MOLM-14, MV-4-11 4 hours,
It is simultaneously hungry 4 hours with the only culture basal cell containing 1%FBS (calf serum), collect sample.Determine compound thin to this three plants
The impact (Fig. 2) of STAT5, C-Myc, ERK, NF- κ B p65, AKT albumen and phosphorylation in born of the same parents.
Experimental result is as shown in Figure 2：In MOLM-14 clones, consumingly can suppress for Buddhist nun (PCI32765) according to Shandong
The phosphorylation of protein kinase FLT3, its EC50 are 0.039 μM.In MOLM-13 and MV-4-11 clones, Buddhist nun is replaced according to Shandong
(PCI32765) phosphorylation of protein kinase FLT3 also can significantly be suppressed, its EC50 is 0.32 μM, 0.36 μM of (figure respectively
2).Additionally, carry FLT3/ITD mutated genes acute myeloid leukemia cells in children MOLM-13, MOLM-14, MV-4-11 this
In three plants of cells, there is strong suppression to the phosphorylation of FLT3/ITD downstreams albumen STAT5 in cell for Buddhist nun (PCI32765) according to Shandong
Make and use, its EC50 is 0.32 μM, 0.11 μM, 0.91 μM respectively, and to the PROTEIN C-Myc closely related with FLT3 protein kinases
There is obvious degradation.In same experiment, control BTK kinase inhibitors AVL-292, CGI1746 concentration be 1 μM when pair
The phosphorylation of protein kinase FLT3 does not have any suppression, and CGI1746 is to FLT3/ITD downstreams albumen STAT5 and albumen in cell
C-Myc also no any impacts.And compare FLT3/ITD kinase inhibitors AC220 consumingly can suppress protein kinase FLT3 and
The phosphorylation and protein degradation C-Myc of Protein S TAT5 closely related with FLT3/ITD.Embodiment 3 shows BTK kinase inhibitions
Agent can suppress the phosphorylation of protein kinase FLT3 according to Shandong for Buddhist nun (PCI32765), affect the signal of protein kinase FLT3 in cell
The phosphorylation of passage downstream Protein S TAT5, and then suppress to carry the acute myeloid leukemia cells in children of FLT3/ITD mutated genes
The cell propagation of strain.
Embodiment 4：Replace Buddhist nun (PCI32765) on cell on apoptotic impact according to Shandong
In order to prove that the death of cell is, by apoptosis or necrosis, to carry FLT3/ITD mutated genes for medication later
Acute myeloid leukemia cells in children MOLM-13, MOLM-14, MV-4-11 and carry FLT3A680V mutated genes acute marrow
Property leukaemia OCI-AML-3, carry wild type FLT3 gene acute myeloid leukemia cells in children NOMO-1 in, have detected according to
Shandong is polymerized to the poly- adenosine diphosphate-ribose of DNA repair enzymes closely related with Apoptosis in cell for Buddhist nun (PCI32765)
The impact of enzyme PARP, 3 protein cleavages of aspartic acid proteolytic enzyme Caspase containing cysteine.With 0 μM of variable concentrations,
0.3 μM, 1 μM, the FLT3 kinase inhibitions that Buddhist nun (PCI32765), 1 μM (in DMSO) are replaced according to Shandong of 3 μM, 10 μM (in DMSO)
Agent AC220,1 μM of BTK kinase inhibitor AVL-292, the BTK kinase inhibitor CGI1746 of 1 μM (in DMSO) are processed respectively
MOLM-13, MOLM-14, MV-4-11, OCI-AML-3, NOMO-1, are then collected behind 12 hours, 24 hours, 48 hours respectively
Cell.Detect that with Western Blot the medicine of variable concentrations is poly- in different time sections adenosine diphosphate-ribose poly- to DNA repair enzymes
The impact of the shear protein of synthase PARP and the aspartic acid proteolytic enzyme Caspase 3 containing cysteine.
Experimental result is as shown in Figure 4：For the acute myeloid leukemia cells in children strain for carrying FLT3/ITD mutated genes
MOLM-13, when being 3 μM for Buddhist nun (PCI32765) Drug level according to Shandong, just it can be seen that significantly DNA is repaiied after acting on 12 hours
The shearing of poly- adenosine diphosphate-ribose polymerase PARP of multiple enzyme, and aspartic acid proteolytic enzyme of the part containing cysteine
The shearing of Caspase 3, similarly can also observe same phenomenon using 1 μM of FLT3 kinase inhibitors AC220, and use
1 μM of BTK kinase inhibitor AVL-292,1 μM of BTK kinase inhibitors CGI1746 then can't see the poly- gland of any DNA repair enzymes
The shearing of glycosides diphosphonic acid-ribose polymerase PARP or the aspartic acid proteolytic enzyme Caspase 3 containing cysteine.For taking
Acute myeloid leukemia cells in children MOLM-14 with FLT3/ITD mutated genes, when according to Shandong replace Buddhist nun (PCI32765) Drug level
For 10 μM when, after acting on 12 and 24 hours respectively, it can be seen that the obvious poly- adenosine diphosphate-ribose polymerase of DNA repair enzymes
The shearing of PARP.For carry FLT3/ITD mutated genes acute myeloid leukemia cells in children MV-4-11, when according to Shandong replace Buddhist nun
(PCI32765) when Drug level is 3 μM, after acting on 12 and 24 hours respectively, it can be seen that the obvious poly- adenosine of DNA repair enzymes
The shearing of diphosphonic acid-ribose polymerase PARP, and aspartic acid proteolytic enzyme Caspase 3 of the part containing cysteine
Shearing.But in the acute myeloid leukemia cells in children OCI-AML-3 for carrying FLT3A680V mutated genes, even if replacing according to Shandong
The Drug level of Buddhist nun (PCI32765) is 10 μM, after acting on 12,24 and 48 hours respectively, does not also see the poly- gland of DNA repair enzymes
The shearing of glycosides diphosphonic acid-ribose polymerase PARP, and aspartic acid proteolytic enzyme Caspase 3 containing cysteine cuts
Cut.In the acute myeloid leukemia cells in children NOMO-1 for carrying FLT3 wild type genes, even if according to Shandong for Buddhist nun's (PCI32765)
Drug level is 10 μM, after acting on 12 and 24 hours respectively, also can not see the poly- adenosine diphosphate-ribose of DNA repair enzymes
The shearing of polymerase PARP, and the shearing of the aspartic acid proteolytic enzyme Caspase 3 containing cysteine；Effect 48 hours
Afterwards, it can be seen that have the shearing of poly- adenosine diphosphate-ribose polymerase PARP of part DNA repair enzymes.Embodiment 4 is demonstrated according to Shandong
The apoptosis of the acute myeloid leukemia cells in children for carrying FLT3/ITD mutated genes can be caused for Buddhist nun (PCI32765).
Embodiment 5：The impact of Buddhist nun (PCI32765) cell cycle on cell is replaced according to Shandong
In order to study which growth cycle cell after medication is stopped in, the acute of FLT3/ITD mutated genes is being carried
The acute myelogenous white blood of marrow leukaemia cell MOLM-13, MOLM-14, MV-4-11 and carrying FLT3 A680V mutated genes
In sick cell OCI-AML-3 cell lines, test according to Shandong for Buddhist nun (PCI32765) to the cell cycle distribution of these cell lines
Affect.With 0 μM of variable concentrations, 0.5 μM, 1 μM (in DMSO) according to Shandong replace Buddhist nun (PCI32765), 1 μM (in DMSO's)
The FLT3 kinase inhibitor AC220 of BTK kinase inhibitors CGI1746 and 0.01 μM (in DMSO) act on three plants of carryings
Acute myeloid leukemia cells in children MOLM-13, MOLM-14, MV-4-11 of FLT3/ITD mutators and one plant of carrying FLT3
The acute casual leukaemia OCI-AML-3 of A680V mutated genes, after acting on 24 hours, collects cell, 1XPBS bufferings
Liquid is washed twice, and 75% ethanol fixes 24 hours in -20 DEG C, and 1XPBS buffer solutions are washed twice again, plus 0.5mL 1XPBS slow
The PI dyeing liquors (purchased from U.S. BD Bioscience) of liquid and 0.5mL are rushed in cell and cell is positioned over into dark lucifuge 37
DEG C dyeing 15 minutes, with flow cytometer (BD FACS Calibur) detect cell cycle distribution.
Experimental result is as shown in Figure 5：In the acute myeloid leukemia cells in children strain MOLM- for carrying FLT3/ITD mutated genes
In 14, with from 0.5 μM increasing to 1 μM for the drug concentration of Buddhist nun (PCI32765) according to Shandong, the cell of the G0-G1 phases of capture also from
54.47% increases to 58.13%；The G0-G1 phases that selective stronger FLT3 kinase inhibitors AC220 can be captured at 0.01 μM
Cell just up to 80.15%；And the cell of the G0-G1 phases of 1 μM of BTK kinase inhibitors CGI1746 capture is only with (only compareing
Plus the DMSO of equivalent, it is not added with any medicine) quite (Fig. 5 a).For the acute myeloid leukaemia for carrying FLT3/ITD mutators
Cell MV-4-11, with from 0.5 μM increasing to 1 μM for Buddhist nun's (PCI32765) drug concentration according to Shandong, the G0-G1 phases of capture it is thin
Born of the same parents also increase to 71.75% from 64.94%；What selective stronger FLT3 kinase inhibitors AC220 can be captured at 0.01 μM
The cell of G0-G1 phases is just up to 97.29%；And the cell of the G0-G1 phases of 1 μM of BTK kinase inhibitors CGI1746 capture only and
Control is quite (Fig. 5 b).For the acute myeloid leukemia cells in children MOLM-13 for carrying FLT3/ITD mutators, with replacing according to Shandong
Buddhist nun's (PCI32765) drug concentration increases to 1 μM from 0.5 μM, and the cell of the G0-G1 phases of capture also increases to from 59.40%
67.41%；The cell of the G0-G1 phases that selective stronger FLT3 kinase inhibitors AC220 can be captured at 0.01 μM is just up to
73.53%；And the cell of the G0-G1 phases of 1 μM of BTK kinase inhibitors CGI1746 capture is only suitable (Fig. 5 c) with control.For
The acute myeloid leukemia cells in children OCI-AML-3 of FLT3 A680V mutated genes is carried, and no matter Buddhist nun (PCI32765) is replaced according to Shandong
Or the cell of the G0-G1 phases of BTK kinase inhibitors CGI1746 captures lacks and control is suitable.For carrying FLT3 A680V
The acute myeloid leukemia cells in children OCI-AML-3 of mutated genes, either according to Shandong replace Buddhist nun (PCI32765) or it is selective compared with
The distribution of strong FLT3 kinase inhibitor AC220 cell cycles does not significantly affect.This is proved in terms of another
According to Shandong for Buddhist nun (PCI32765) for the acute myeloid leukemia cells in children for carrying FLT3/ITD mutators has significantly effect,
And the distribution of cell cycle also has significantly affects (Fig. 5 d).For the acute myelogenous white blood for carrying FLT3 wild type genes
Sick cell NOMO-1, either replaces Buddhist nun (PCI32765) or selective stronger FLT3 kinase inhibitor AC220 to thin according to Shandong
The distribution in born of the same parents' cycle does not significantly affect.This more demonstrates prominent for FLT3/ITD is carried for Buddhist nun (PCI32765) according to Shandong
The acute myeloid leukemia cells in children for becoming gene has significantly effect, and the distribution of cell cycle also has significantly impact (figure
5e)。
Claims (7)
1. preparing for treating the white blood of acute myeloid for carrying FLT3/ITD mutated genes for Buddhist nun (PCI32765) according to Shandong
Purposes in the medicine of patient.
2. purposes according to claim 1, wherein be administered alone for Buddhist nun according to Shandong or with one or more other treatment
Agent is administered in combination.
3. it is a kind of suppress carry FLT3/ITD mutated genes Acute Myeloid Leukemia Cells Contributing non-treatment method, including by
The cell with contact for Buddhist nun according to Shandong.
4. method according to claim 3, wherein the white blood of acute myeloid of the carrying FLT3/ITD mutated genes
Sick cell is one or more in MOLM-13, MOLM-14, MV-4-11, PL-21 and MUTZ-11.
5. the method according to claim 3 or 4, wherein the cell and valid density are at least 0.05 μM replacing according to Shandong
Buddhist nun contacts.
6. the medicine of the Acute Myeloid Leukemia Cells Contributing of FLT3/ITD mutated genes is carried for suppression in preparation for Buddhist nun according to Shandong
Purposes in thing.
7. purposes according to claim 6, wherein the white blood of acute myeloid of the carrying FLT3/ITD mutated genes
Sick cell is one or more in MOLM-13, MOLM-14, MV-4-11, PL-21 and MUTZ-11.
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