CN105816467A - Application of Etonogestrel in preparation of products for preventing B-cell lymphoma - Google Patents
Application of Etonogestrel in preparation of products for preventing B-cell lymphoma Download PDFInfo
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- CN105816467A CN105816467A CN201610156842.5A CN201610156842A CN105816467A CN 105816467 A CN105816467 A CN 105816467A CN 201610156842 A CN201610156842 A CN 201610156842A CN 105816467 A CN105816467 A CN 105816467A
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- etonogestrel
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- cell lymphoma
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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
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
- A61K31/567—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in position 17 alpha, e.g. mestranol, norethandrolone
Abstract
The invention discloses an application of Etonogestrel in the preparation of products for preventing B-cell lymphoma. The invention provides an application of Etonogestrel in the preparation of products for treating B-cell lymphoma. The experiment results show that Etonogestrel, which has been certified by FDA and CFDA, is repositioned as an antitumor drug; according to the different cell lines (tissue types) of tumor and mutant sites, the drugs, which are not used to treat tumors, are screened, the inventor finds that Etonogestrel can prevent B-cell lymphoma, and a novel application of Etonogestrel is found.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of Etonogestrel application in preparing anti-B cell lymphoma product.
Background technology
Tumor be threaten the most common of human health be also the most serious a kind of disease, research and development high efficiency anti-tumor medicine to extend patient life cycle most important.In recent years, along with developing rapidly of cancer genomics and molecular pharmacology, the research and development of new type antineoplastic medicine achieve good achievement, but owing to new drug development puts into big, the bottlenecks such as cycle length cannot overcome, and the feature such as tumor individual inheritance variation is big, causes many traditional anti-tumor medicine effects to be not added with, new drug is expensive, and side effect is the brightest.
Etonogestrel is a kind of subcutaneous implant being mainly used in contraception, belongs to steroid progestogen.The contraception mechanism of this medicine is main by realizing the ovulation inhibitory action of pituitary-gonadal axis, can also increase the sliminess of cervical mucus simultaneously, prevent the entrance of sperm.Currently also there is people, at dorsal hippocampus injection Etonogestrel, dementia rats learning and memory influencing mechanism is carried out medicine reorientation research.
Summary of the invention
This research is exactly based on the cancer gene of integration cancer group Cosmicversion72 data base and composes the drug data base of FDA approval in CancerGeneCensus and protein interaction STRINGversion10 data base and DrugBankversion4.2, the candidate's reorientation medicine obtained, tumor cell line is carried out examination experiment, the antitumor drug that screening makes new advances.The candidate tumor suppression medicine doing tumor cell line screening is shown in as follows:
Nicardipine,Promethazine,Estrone,Etonogestrel,Sulindac,Etonogestrel,Etonogestrel,Levonorgestrel,Mesalazine,Indomethacin,Sulfasalazine,Balsalazide,Irbesartan,Ibuprofen,Isoprenaline,PentosanPolysulfate。
It is an object of the present invention to provide the new application of Etonogestrel.
The Etonogestrel that the present invention provides application in preparation treatment B cell lymphoma product.
Second object of the present invention is to provide the new application of Etonogestrel.
The invention provides Etonogestrel application in preparation suppression B cell lymphoma cell proliferation product.
Third object of the present invention is to provide the new application of Etonogestrel.
The invention provides Etonogestrel application in preparation reduces B cell lymphoma cell IC50 value product.
In above-mentioned application, described B cell lymphoma cell is BC-3.
In above-mentioned application, described product is medicine or test kit.
Fourth object of the present invention is to provide a kind of product.
The product that the present invention provides, its active component is Etonogestrel；Described product has following 1)-3) at least one function:
1) treatment B cell lymphoma；
2) suppression B cell lymphoma cell proliferation；
3) B cell lymphoma cell IC50 value is reduced.
In the said goods, described B cell lymphoma cell is BC-3.
In the said goods, described product is medicine or test kit.
The experiment proves that, the present invention is to FDA, medicine Etonogestrel the most granted for CFDA carries out tumour medicine reorientation, the cell line (organization type) different according to tumor and mutational site are not that anti-tumor drug screens to indication, find Etonogestrel this new application of anti-B cell lymphoma, it is achieved old medicine is newly used.
Accompanying drawing explanation
Fig. 1 is 96 well culture plate medicine sieve medicine template distributions.
Fig. 2 is that Etonogestrel is sensitive to B cell lymphoma；EC50=9.3750；IC50=6.5544；R2=0.9991.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
In following embodiment, medicine to be measured is Etonogestrel, and its chemical structural formula is:
It is drugbank product, and catalog number is DB00294.
The source of B cell lymphoma cell BC-3, people's Lung Squamous Carcinoma Cells NCI-H1703 and human lung carcinoma cell NCI-H2122 in example below is as follows:
BC-3ATCCCRL-2277
NCI-H1703ATCCCRL-5889
NCI-H2122ATCCCRL-5985
Main instrument in example below and consumptive material
DMSO(fromSigma,Cat.No.D4540)
Saturating Tissue Culture Plate (fromCorning, Cat.No.3610) at the bottom of 96-well white
CellTiterGlo test kit (fromPromega, Cat.No.G7573)
Doxorubicin positive drug (fromMCE, Cat.No.HY-15142)
FetalBovineSerum(fromGibco,Cat#10099141)
100mm culture dish (fromCorning, Cat#430167)
RPMI-1640medium(fromGibco,Cat#A1049101)
DMEMmedium(fromGibco,Cat#11995081)
DMEM/F12medium(fromGibco,Cat#11330057)
EMEMmedium(fromGibco,Cat#10370021)
Mutidrop384 cell liquor separator (Thermo, Cat#5840150)
The multi-functional plate reading machine of EnSpire (PerkinElmer, Cat#2300-001M)
Embodiment 1, CELLTITER-GLO detect the anti-B cell lymphoma of Etonogestrel
One, the preparation of orifice plate to be measured
1, plating cells
A) complete medium needed for each cell is prepared.
B) before experiment starts, confirmation flag medicine sieve cell name on 100mm culture dish, culture medium and generation time, the information such as generation, it is ensured that test errorless.
C) attached cell operation is with reference to step d) to i), and suspension cell operation reference step j) is to l).
D) vacuum pump is utilized to draw cell culture medium during sterile working.
E) with the aseptic PBS solution rinse cell surface layer of 2ml, then with vacuum pump sucking-off PBS waste liquid.
F) 1ml0.25% (w/v) Trypsin-0.038% (w/v) EDTA solution digestion cell is added gently to culture dish, mix gently several under, solution covers cell surface layer, observation of cell digestion situation under inverted microscope, terminates trypsinization effect when cell will come off.
G) in culture dish, add 5ml37 DEG C of preheated complete medium, blow and beat cell gently with pipet so that it is split away off bottom culture dish.
H) transferring in 15ml or 50ml sterile centrifugation tube by this cell suspension, 1000rpm is centrifuged 5min.
I) vacuum pump sterile working sucking-off supernatant culture medium is utilized.Add 5ml37 DEG C of preheated complete medium re-suspended cell precipitation, blow and beat mixing gently.
J) cell is blown and beaten gently with pipet so that it is completely fall off bottom culture dish.
K) transferring in 15ml or 50ml sterile centrifugation tube by this cell suspension, 1000rpm is centrifuged 5min.
L) vacuum pump sterile working sucking-off supernatant culture medium is utilized.Add 5ml37 DEG C of preheated complete medium re-suspended cell precipitation, blow and beat mixing gently.
M) with cell counter, cell suspension is counted, adjust cell suspension and carry out plating cells experiment to proper density fishplate bar.
BC-3 cell is carried out according to the method described above, obtains BC-396 porocyte culture plate.
BC-3 cell, complete medium is RPMI-1640, and for life product, A1049101, fishplate bar density (cells/well) is 4000.
2, medicine Etonogestrel to be measured preparation and dosing (200X final concentration):
1) medicine Etonogestrel motherboard to be measured preparation
A) with DMSO, medicine Etonogestrel to be measured is diluted to 20mM stand-by.
Take the 20mM medicine to be measured 79 μ L configured in a) step and add in dilution plate the first row the first hole, be subsequently added the DMSO solvent of 54 μ L to the first row the second hole to the 9th hole in, take in 25 μ L solution to the second hole from the first hole, take in 25 μ L solution to the 3rd hole after mixing from the second hole, the like to the 9th hole, it is ensured that medicine gradually carries out 3.16 times of multiple proportions gradient dilutions.
2) positive drug Doxorubicin (MCE, Cat.No.HY-15142) motherboard preparation
A) with DMSO, positive drug Doxorubicin is diluted to 6mM stand-by.
B) 6mM positive drug Doxorubicin solution is added in dilution plate, DMSO solution this medicine to be measured of 1:3.16 times of multiple proportions gradient dilution.
3, the preparation of medicine working plate and dosing
A) medicine to be measured and positive drug sample-adding template are illustrated in fig. 1 shown below, wherein, S1208: positive drug Doxorubicin, DMSO: Positive control wells, Cpd1, final concentration of 0.5% (DMSO is compatible) of 2,3: medicine to be measured, DMSO.
B) in working plate, add the 95 μ specific complete mediums of l cell, corresponding 9 holes of each medicine, with multiple tracks volley of rifle fire difference transferase 45 μ l a series of (9 holes) solution (10X final concentration) that doubling dilution is good the most successively from medicine to be measured and positive drug doxorubicin motherboard, obtain the cell culture medium containing variable concentrations medicine.
C) from incubator, take out step 1 and prepare BC-396 porocyte culture plate, in BC-396 porocyte culture plate, the 10 above-mentioned b of μ l are added respectively by Fig. 1 dosing template arrangement mode (Fig. 1)) cell culture medium (10X final concentration) containing variable concentrations medicine prepared, put into CO2Incubator 37 DEG C cultivates 72h, obtains the 96 hole medicine sieve plates of BC-3.
Using be not added with any medicine as control wells.
Final concentration in 96 orifice plates of above-mentioned medicine to be measured, positive drug Doxorubicin and control wells and dosing situation are as follows:
The medicine to be measured final concentration (μM) in the 2-10 hole of Fig. 1 is followed successively by: 100,31.64557,10.01442,3.16912,1.002886,0.317369,0.100433,0.031783,0.010058；
Positive drug Doxorubicin final concentration (μM) in the 2-10 hole of Fig. 1 is followed successively by: 30,9.493671,3.004326,0.950736,0.300866,0.095211,0.03013,0.009535,0.003017；
In addition, S1208 hole (E1-H1 and A12-D12): 10 100 μMs of Doxorubicin solution of μ l final concentration (solvent is the complete medium solution comprising 0.5%DMSO) in 96 orifice plates, DMSO hole (A1-D1, E12-H12 and A11-H11): 10 μ l comprise the complete medium solution of 0.5%DMSO.
Two, CELLTITER-GLO fluorecyte activity monitor system detection
1, CellTiter-Glo preparation of reagents
A) before using, CellTiter-Glo reagent Buffer is melted, equilibrate to room temperature and use.
B) before using, CellTiter-Glo reagent agar substrate is melted, equilibrate to room temperature and use.
C) take the CellTiter-GloBuffer that 100ml balances and join in bottled CellTiter-Glo reagent agar substrate so that it is abundant resuspended formation enzyme/substrate mixture, the most so-called CellTiter-Glo detectable.
D) mix vortex gently, and be repeatedly inverted the uniform solution of acquisition.In general, in 1 minute, CellTiter-Glo substrate reagent will fully dissolve, subpackage, keeps in Dark Place standby at-20 DEG C, it is to avoid multigelation.
2, detection
A), before detection, the 96 hole medicine sieve plates of the 3 of above-mentioned one BC-3 obtained are balanced 20-30 minute at room temperature.
B) cellular morphology and the death condition of every piece of culture plate observed by inverted microscope, marks abnormal conditions repetition measurement once.
C) in all medicine sieve apertures, add the CellTiter-Glo reagent of above-mentioned 1 preparation of 100 μ l, mixing.
D) on 96 hole microwell plate agitators, fully concussion mixes 2 minutes, makes cell crack completely.
E) lucifuge room temperature carries out luminescence signal detection after placing 15 minutes, it is ensured that the stability of signal.
Luminescence signal is read when f) using EnSpire multi-functional plate reading machine 570nm.
G) analyzing and processing data.
The 96 hole medicine sieve plate results of BC-3 are as shown in Figure 2.
Calculating IC50 value, result is as shown in table 1.
Same method detection Etonogestrel is used to make the IC50 value of employment Lung Squamous Carcinoma Cells NCI-H1703 and human lung carcinoma cell NCI-H2122, result such as table 1.
It can be seen that Etonogestrel has Specific Inhibitory Effect to B cell lymphoma cell proliferation, can be used to the medicine as treatment B cell lymphoma.
Table 1 is different cell IC50 values under Etonogestrel medicine effect
| Cell | IC50 value |
| BC-3 | 6.5544 |
| NCI-H1703 | 100 |
Claims (7)
1.Etonogestrel application in preparation treatment B cell lymphoma product.
2.Etonogestrel application in preparation suppression B cell lymphoma cell proliferation product.
Application the most according to claim 1 and 2, it is characterised in that: described B cell lymphoma cell is BC-3.
4. according to described application arbitrary in claim 1-3, it is characterised in that: described product is medicine or test kit.
5. a product, its active component is Etonogestrel；Described product has following 1) and/or 2) function:
1) treatment B cell lymphoma；
2) suppression B cell lymphoma cell proliferation.
Product the most according to claim 5, it is characterised in that: described B cell lymphoma cell is BC-3.
7. according to the product described in claim 5 or 6, it is characterised in that: described product is medicine or test kit.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610156842.5A CN105816467B (en) | 2016-03-18 | 2016-03-18 | Etonogestrel is preparing the application in anti-B cell lymphoma product |
| PCT/CN2016/094103 WO2017156967A1 (en) | 2016-03-18 | 2016-08-09 | Use of etonogestrel in preparation of anti-b cell lymphoma products |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610156842.5A CN105816467B (en) | 2016-03-18 | 2016-03-18 | Etonogestrel is preparing the application in anti-B cell lymphoma product |
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| Publication Number | Publication Date |
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| CN105816467A true CN105816467A (en) | 2016-08-03 |
| CN105816467B CN105816467B (en) | 2019-01-22 |
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|---|---|---|---|---|
| WO2017156967A1 (en) * | 2016-03-18 | 2017-09-21 | 上海交通大学 | Use of etonogestrel in preparation of anti-b cell lymphoma products |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105012314A (en) * | 2015-06-30 | 2015-11-04 | 上海交通大学 | Application of Etonogestrel in the preparation of anti-prostate cancer products |
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| CN105816467B (en) * | 2016-03-18 | 2019-01-22 | 上海交通大学 | Etonogestrel is preparing the application in anti-B cell lymphoma product |
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| CN105012314A (en) * | 2015-06-30 | 2015-11-04 | 上海交通大学 | Application of Etonogestrel in the preparation of anti-prostate cancer products |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017156967A1 (en) * | 2016-03-18 | 2017-09-21 | 上海交通大学 | Use of etonogestrel in preparation of anti-b cell lymphoma products |
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| CN105816467B (en) | 2019-01-22 |
| WO2017156967A1 (en) | 2017-09-21 |
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