CN106943400A - Strengthen compound and its preparation that liver cancer cells kill sensitiveness to CIK cell - Google Patents
Strengthen compound and its preparation that liver cancer cells kill sensitiveness to CIK cell Download PDFInfo
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- CN106943400A CN106943400A CN201710290463.XA CN201710290463A CN106943400A CN 106943400 A CN106943400 A CN 106943400A CN 201710290463 A CN201710290463 A CN 201710290463A CN 106943400 A CN106943400 A CN 106943400A
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- 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
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
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Abstract
Strengthen compound and its preparation that liver cancer cells kill sensitiveness to CIK cell the invention discloses a kind of, specifically provide a kind of application of pyridine-imidazole class organic compound in preparing enhancing liver cancer cells to kill CIK cell the medicine of sensitiveness.Present invention demonstrates that pyridine-imidazole class compound can effectively strengthen liver cancer cells kills sensitiveness to CIK cell, pharmaceutically acceptable pharmaceutical preparation can be prepared into, just play preferably immunotherapeutic effects using a small amount of CIK cell to realize.
Description
Technical field
The invention belongs to immunotherapy field, it is related to CIK cell, and in particular to one kind enhancing liver cancer cells are to CIK cell
Kill the compound and its preparation of sensitiveness.
Background technology
Primary carcinoma of liver (hereinafter referred to as liver cancer) is one of most common malignant tumour in world wide, every year about 63
Ten thousand new cases, and the incidence of disease is on the rise in recent years.The course of disease of liver cancer patient is short, clinically more difficult early detection,
Often the state of an illness has been enter into middle and advanced stage when making a definite diagnosis, and case fatality rate is high.The treatment of current liver cancer is mainly with surgery excision, liver transfer operation, office
Portion's ablation, chemotherapy embolism and other locoregional treatments, molecular targeted therapy etc., but these methods therapeutic effect not
Ideal, because the innate immunity, specific immune function are low in liver cancer patient body, radical surgery resection rate is low, and Post operation is multiple
Hair rate is high, radiation, chemotherapy weak curative effect, easily occurs to send out and extrahepatic metastases in liver, therefore is badly in need of finding new effective ways
To treat liver cancer.Biological therapy has become the 4th kind of pattern in combined therapy of tumour, is increasingly taken seriously.
Mainly there are tumor infiltrating lymphocyte (TIL), dendron currently used for the immunocompetent cell of tumor biotherapy
Shape cell (DC) and cytokine induced kill cell (CIK) etc..CIK cell is a kind of new immunocompetent cell,
The surface marker of its main effects cell is CD3+CD56+, compared with other adoptive immunotherapy cells, with growth rate
It hurry up, kill the advantages of tumor activity is high, kill knurl composes wide.Research shows, adoptive cellular immunotherapy to suppress liver cancer recurrence and transfer,
Raising patient cure rate, improvement patients ' life quality, extension life cycle all play an important roll.
How an important research that preferably immunotherapeutic effects be researcher is just played with less CIK cell
Direction.
The content of the invention
Strengthen compound and its system that liver cancer cells kill sensitiveness to CIK cell it is an object of the invention to provide a kind of
Agent, just to play preferably immunotherapeutic effects using less CIK cell.
Realize that above-mentioned purpose technical scheme of the present invention is as follows：
A kind of pyridine-imidazole class organic compound kills the medicine of sensitiveness preparing enhancing liver cancer cells to CIK cell
In application, the pyridine-imidazole class organic compound has below formula structure：
Wherein, R2For-OCH2CH3Or-OH；
R1For-CH2- or-CH (CH3)2- or-CH2CH2CH2-。
Preferably, pyridine-imidazole class organic compound is selected from following compounds：
It is a kind of to be used to strengthen the pharmaceutical preparation that liver cancer cells kill CIK cell sensitiveness, including above-mentioned pyridine-imidazole
Class organic compound, also including pharmaceutically acceptable carrier or excipient, is made pharmaceutically acceptable formulation.
Preferably, the pharmaceutically acceptable carrier or excipient include one or more solids, semi-solid or liquid
Body auxiliary material.
Preferably, the pharmaceutically acceptable formulation include tablet, capsule, granule, injection, pill, dissipate
Agent, paste, oral liquid.
The outstanding advantages of the present invention：
Present invention demonstrates that pyridine-imidazole class compound can effectively strengthen liver cancer cells kills sensitiveness to CIK cell,
Pharmaceutically acceptable pharmaceutical preparation can be prepared into, is controlled with realizing just to play preferably to be immunized using a small amount of CIK cell
Therapeutic effect.
Brief description of the drawings
Fig. 1 compares HepG2 killing rate for each group CIK cell.
Embodiment
The just in conjunction with the embodiments specific essentiality content for introducing the present invention below, due to length reason, experimentation is retouched
Stating can not accomplish very in detail, and the part not being described in detail in every experiment is conventional behaviour well known to those skilled in the art
Make.
First, experiment material
Numbering CM1-6 pyridine-imidazole class organic compound is known compound, and personnel's reference is synthesized by our company
Document and the synthesis of organic synthesis conventional method, and confirmed through mass spectrum and nuclear-magnetism.
Human hepatoma cell line HepG2 preserves for a long time for our company.Recombinant human interferon alpha 2 rhIFN- γ, combining human interleukins
Plain rhIL-2, CD3 mouse anti-human monoclonal antibody, CIK cell separating liquid, PBS, serum free medium MD-CM-STMN,
RPMI1640 culture mediums, hyclone FBS, MTT, DMSO, pancreatin are purchased from Nanjing and build up biology.
2nd, experimental method
1st, prepared by PMNC
Aseptic aspiration healthy human peripheral blood about 50mL, anticoagulant heparin (heparin 15IU/mL) dilutes periphery with isometric PBS
Blood.By 1：1 volume ratio is slowly added into the 50mL centrifuge tubes containing cell separation liquid, 2000r/min centrifugations 20min.Take
More intermediate annular milky mononuclearcell layer, is moved into 50mL centrifuge tubes, is added appropriate PBS, 1800r/min centrifugation 8min, is abandoned
Supernatant, is washed 2 times, cell count.
2nd, CIK cell Fiber differentiation
It is 1 × 10 with the MD-CM-STMN cell culture mediums adjustment mononuclearcell density containing 5%FBS6Individual/mL, connects
20mL is planted in blake bottle, TCS is 2 × 107, (the 0th day) same day addition rhIL-21000U/mL, rhIFN- γ
1000U/mL, CD3 mouse anti-human monoclonal antibody 5mg/L, place 37 DEG C, 5%CO2Start culture in incubator.Start within 3rd day to mend
Plus MD-CM-STMN cell culture mediums, while supplementing CD3 mouse anti-human monoclonals antibody, rhIFN- γ, rhIL-2, consumption is the same,
Hereafter fresh culture is added per 3d, rhIFN- γ are added, consumption is identical, rhIL-2 consumptions halve as 500U/mL.
3rd, CIK cell proliferation assay
Trypan Blue method, is counted, weight with cell counting count board respectively at culture the 0th, 5,8,13,20 days to cell
Answer 3 times and average.
4th, the culture of tumor cell line
HepG2 cells are inoculated in blake bottle, the RPMI 1640 containing 10%FBS are added, attached cell uses 0.25%
Pancreatin digestion.Take the logarithm the cell in growth period, be inoculated in 96 orifice plates, carry out killing experiments in vitro.
5th, mtt assay determines CDCC of the CM 1-6 compounds to HepG2 cells
Take the logarithm growth period HepG2 cell, adjustment cell suspension density is 5 × 103Individual/mL, is inoculated in 96 orifice plates, per hole
200μL.After changing liquid after cell attachment, administration group is separately added into 2 μM of compound CM 1-6, and control group adds nutrient solution, and every group equal
If 3 parallel holes, 37 DEG C, 5%CO are placed on2Acted in incubator after 48h, each administration group and control group are respectively added per hole
20 μ LMTT (5g/L), continue to be placed on 37 DEG C, 5%CO24h is cultivated in incubator, supernatant is abandoned, 200 μ L DMSO are added per hole,
Shaking table vibrates 10min at 37 DEG C, and ELIASA determines absorbance (A) value at 490nm.
6th, mtt assay determines CIK to HepG2 killing functions of immunocytes
Using culture to the CIK cell of the 13rd day as effector cell, killing experiments in vitro is carried out.Adjust HepG2 cells dense
Degree 5 × 103Individual/mL, per the μ L of hole 100, is divided into the enhanced sensitivity groups of CM 1, the enhanced sensitivity groups of CM 2, the enhanced sensitivity groups of CM 3, the enhanced sensitivity groups of CM 4, CM 5
Enhanced sensitivity group, the enhanced sensitivity groups of CM 6 and conventional group, CM 1-6 enhanced sensitivities groups add 2 μM of compound CM 1-6 respectively, and conventional group is normal to cultivate,
It is placed on 37 DEG C, 5%CO2Cultivated in incubator and liquid is changed to cell attachment, compare 10 by effect target：Effector cell is added culture by 1
Kong Zhong, while it is right as feminine gender separately to set independent target cell (target cell+nutrient solution) and individual effect cell (effector cell+nutrient solution)
According to being placed on 37 DEG C, 5%CO2Acted in incubator after 48h, add MTT (5g/L), 20 μ L/ holes, continue to be placed on 37 DEG C,
5%CO24h is cultivated in incubator, 2000r/min centrifugation 5min, turnover panel abandons supernatant, adds the μ L/ holes of DMSO 150, and concussion is waited to sink
Form sediment and dissolve, be put in ELIASA, killing activity is calculated as follows in 490nm densitometrics (OD) value：Killing rate (%)=
{ 1- [(experimental group OD values-individual effect cell OD values)/independent target cell OD values] } × 100%.
7th, statistical method
Analyzed using the statistical softwares of SPSS 19.0, data are represented with mean ± standard deviation, P ＜ 0.05 anticipate with statistics
Justice.
3rd, experimental result
1st, CIK cell is bred
CIK cell is accelerated in the 5th day cell proliferation rate of culture, and the 5-13 days were the fast breeding period, at the 20th day
Cell propagation reaches 118 times of former plantation amount, and 176 times of former plantation amount are reached in the 20th day cell propagation.
2nd, CDCCs of the compound CM 1-6 to HepG2 cells
Compound CM 1-6 administration groups and absorbance (A) value of control group are without significant difference, it was demonstrated that 2 μM of compound CM 1-6
To HepG2 cells without obvious CDCC.It the results are shown in Table 1.
The ratio of the absorbance of the compound CM 1-6 administration groups of table 1 and control group
| CM 1 | CM 2 | CM 3 | CM 4 | CM 5 | CM 6 | |
| A administrations/A controls | 1.02 | 0.98 | 0.98 | 0.99 | 1.01 | 1.01 |
3rd, compound CM 1-6 strengthen liver cancer cells to CIK cell killing sensitiveness
Each enhanced sensitivity group CIK cell, which is can be seen that, from table 2 and Fig. 1 to the killing rate of HepG2 cells is all remarkably higher than routine
Group.Known 2 μM of compound CM 1-6 are to HepG2 cells without obvious CDCC, and each enhanced sensitivity group CIK cell is to HepG2 cells
The raising of killing rate can only be attributed to compound CM 1-6 and improve killing sensitiveness of the HepG2 cells to CIK cell, use change
After compound CM 1-6 are to target cell HepG2 cell enhanced sensitivities, effect target is than in the case of constant, killing of the CIK cell to HepG2 cells
Power is significantly improved.
(effect target compares 10 to killing rate of each group CIK cell of table 2 to HepG2 cells:1)
Present invention demonstrates that pyridine-imidazole class compound can effectively strengthen liver cancer cells kills sensitiveness to CIK cell,
Pharmaceutically acceptable pharmaceutical preparation can be prepared into, is controlled with realizing just to play preferably to be immunized using a small amount of CIK cell
Therapeutic effect.
Above-described embodiment is the embodiment to essentiality content of the present invention, for preferably explaining the present invention, but this area skill
Art personnel are it is to be understood that protection scope of the present invention should not be confined to above-mentioned specific embodiment.
Claims (5)
1. a kind of pyridine-imidazole class organic compound is in medicine of the enhancing liver cancer cells to CIK cell killing sensitiveness is prepared
Application, it is characterised in that the pyridine-imidazole class organic compound has below formula structure：
Wherein, R2For-OCH2CH3Or-OH；
R1For-CH2- or-CH (CH3)2- or-CH2CH2CH2-。
2. application according to claim 1, it is characterised in that pyridine-imidazole class organic compound is selected from following chemical combination
Thing：
3. a kind of be used to strengthen the pharmaceutical preparation that liver cancer cells kill CIK cell sensitiveness, it is characterised in that：Will including right
The pyridine-imidazole class organic compound described in 1 or 2 is sought, also including pharmaceutically acceptable carrier or excipient, medicine is made
Acceptable formulation on.
4. pharmaceutical preparation according to claim 3, it is characterised in that：The pharmaceutically acceptable carrier or excipient
Including one or more solids, semisolid or Auxiliary Liquid Material.
5. pharmaceutical preparation according to claim 3, it is characterised in that：The pharmaceutically acceptable formulation includes piece
Agent, capsule, granule, injection, pill, powder, paste, oral liquid.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710290463.XA CN106943400B (en) | 2017-04-28 | 2017-04-28 | Enhance liver cancer cells to the compound and its preparation of CIK cell killing sensibility |
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| CN201710290463.XA CN106943400B (en) | 2017-04-28 | 2017-04-28 | Enhance liver cancer cells to the compound and its preparation of CIK cell killing sensibility |
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| CN106943400B CN106943400B (en) | 2019-09-24 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103626769A (en) * | 2013-12-23 | 2014-03-12 | 山东大学 | Substituted sulfydryl hexahydric heteroaromatic imidazole derivative and preparation method and application thereof |
| CN106083847A (en) * | 2016-08-03 | 2016-11-09 | 山东大学 | A kind of imidazopyridine mercapto phenylacetic acid derivative and preparation method and application |
-
2017
- 2017-04-28 CN CN201710290463.XA patent/CN106943400B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103626769A (en) * | 2013-12-23 | 2014-03-12 | 山东大学 | Substituted sulfydryl hexahydric heteroaromatic imidazole derivative and preparation method and application thereof |
| CN106083847A (en) * | 2016-08-03 | 2016-11-09 | 山东大学 | A kind of imidazopyridine mercapto phenylacetic acid derivative and preparation method and application |
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| Publication number | Publication date |
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| CN106943400B (en) | 2019-09-24 |
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Effective date of registration: 20190828 Address after: 050000 First Floor of Zhitong Pharmaceutical Valley Science Park, 197 Taihang South Street, Shijiazhuang High-tech Zone, Hebei Province Applicant after: Hebei Wanma Biomedical Co., Ltd. Address before: 211198 No. 18 Zhilan Road, Science Park, Jiangning District, Nanjing City, Jiangsu Province Applicant before: Nanjing cover Seef Pharmaceutical Technology Co. Ltd. |
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