CN106075453A - A kind of anti-tumor medicinal preparation combination - Google Patents
A kind of anti-tumor medicinal preparation combination Download PDFInfo
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Abstract
The present invention provides the combination of a kind of anti-tumor medicinal preparation, and it includes IFN γ and IDO inhibitor, or IFN γ and AhR inhibitor.The tumor cell of differentiation not only can be killed by the anti-tumor medicinal preparation combination that the present invention provides, and can be killed by the tumor cell that enter dormancy, has the effect thoroughly removing tumor cell.
Description
Technical field
The present invention relates to a kind of pharmaceutical preparation combination, particularly relate to a kind of anti-tumor medicinal preparation combination.
Background technology
Malignant tumor is as the disease of a kind of serious threat human life health, and searching in recent years is effective, toxic and side effects is little
Therapeutic Method become the most important thing of medical research.
The Biotherapeutics of tumor is the fourth-largest oncotherapy technology after operation, radiation and chemotherapy, and it uses biology
Immunocyte to gathering from the patient of technology and biological preparation or immune factor feed back to after carrying out In vitro culture and amplification
Method in patient body, excites, enhancing body autoimmune function, reaches to treat the purpose of tumor.Its safety is high, kill tumor
High specificity, the advantage having no side effect, be referred to as " the green bio therapy " of tumor subject.
Tumor dormancy (tumordormancy) is a kind of phenomenon the most generally existed, and is also malignant cell
One of biological property, the long-term existence of rest cell is the main cause that malignant tumor is difficult to thoroughly effect a radical cure, and is also to cause swelling
One of root of tumor recurrence and metastasis.
Owing to recognizing unclear to the mechanism of Tumor dormancy, use the method treating tumor at present, although can temporarily realize
Healing to tumor clinically, but owing to the tumor cell of dormancy is not effectively killed, after some moons or year, tumor again can
Recurrence or transfer.
Therefore, how to provide and can be killed by the tumor cell of dormancy, reaching thoroughly to remove the effect of tumor becomes and needs
The problem solved.
Summary of the invention
The invention provides the combination of a kind of anti-tumor medicinal preparation, including IFN-γ and IDO inhibitor, or IFN-γ with
AhR inhibitor, is used in combination above-mentioned preparation and not only can be killed by the tumor cell of differentiation, and can will enter the swollen of dormancy
Oncocyte is killed, and has the effect thoroughly removing tumor cell.
In the scheme of the application, described IFN-γ and IDO inhibitor, or IFN-γ and AhR inhibitor, claim as entirety
Make the anti-tumor medicinal preparation combination of the present invention.
In the scheme of the application, IFN-γ can be commercially available IFN-γ preparation, or by adenovirus vector
The IFN-γ preparation carried；IDO inhibitor includes various competitiveness and noncompetitive indoleamine 2, and 3-is double adds oxidase inhibitor
(indoleamine 2,3-dioxygenase) inhibitor；AhR inhibitor includes various aromatic hydrocarbon receptor (arylhydrocarbon
Receptor) completely or partially antagonist.
In a specific embodiment of the present invention, described IDO inhibitor includes 1-methyl tryptophan (1-methyl-
Tryptophan), it is called for short 1-MT.
Further, described AhR inhibitor includes dimethylformamide, is called for short DMF.
Further, IFN-γ, IDO inhibitor and AhR inhibitor in the combination of described anti-tumor medicinal preparation are
Unit formulation.
IFN-γ, IDO inhibitor and AhR inhibitor in the described anti-tumor medicinal preparation combination of the present invention are unit
Preparation.Described unit formulation is to meet the preparation of effective ingredient needed for single administration, such as a unit (pin) injection etc..Patient is once
The amount using required medicine can be conveniently by unit bodies heavy prescription needed for the body weight of calculating patient and this patient's a drug
The product of amount obtains.Such as, during preparing medicine, it is considered that adult's body weight is 50-70kg, can be dynamic by experiment
Dose,equivalent conversion relation between the per weight dosage of thing and people determines dosage.For example, it is possible to according to FDA, SFDA
The instruction proposed Deng medicine administrative organ, it is possible to reference to (Huang continues the Chinese etc., " in pharmacological testing between animal and animals and human beings body
Between dose,equivalent conversion ", " Chinese Clinical pharmacology and therapeutics ", 2004Sep；9 (9): 1069-1072) determine.At this
In the embodiment of invention, it is possible to use convert people and mice according to the body surface area conversion factor 0.0026 of people and mice
Dosage.
In the solution of the present invention, being the mice of 20g for body weight, IFN-γ is with 5 × 104-1×105The amount of U is administered to
Mice, IDO inhibitor is administered to mice with the amount of 15-20mg and (such as the mice of 20g, is formulated as by IDO inhibitor water
The solution of 5mg/mL, mice gavages this solution 3-4mL every day), AhR inhibitor is used with the amount of 5mg/kg Mouse Weight.
Further, according to the experiment for mice, for the described IFN-γ unit of people's (such as 50-70kg body weight)
Preparation includes 1 × 107～4 × 107The IFN-γ of U；Described IDO inhibitor unit formulation includes the IDO inhibitor of 4～8g；
Described AhR inhibitor includes the AhR inhibitor of 27-50mg.
Further, described IDO inhibitor is 1-MT.
Further, described AhR inhibitor is DMF.
The administering mode of the anti-tumor medicinal preparation combination that the present invention provides is: use described antitumor to suffering from tumor individuality
Pharmaceutical preparation is combined, such as, vein, intraperitoneal or intratumor injection IFN-γ, Combined with Oral or intratumor injection IDO inhibitor；
Or vein, intraperitoneal or intratumor injection IFN-γ, associating gavage or intratumor injection AhR inhibitor.
Present invention also offers a kind for the treatment of or the method for prophylaxis of tumours, including suffering from the individuality of tumor or there is tumor sending out
The individuality of raw trend uses the process of described anti-tumor medicinal preparation combination.Detailed process is note in vein, intraperitoneal or tumor
Penetrate IFN-γ, Combined with Oral or intratumor injection and be administered IDO inhibitor；Or vein, intraperitoneal or intratumor injection IFN-γ, connection
Close gavage or intratumor injection is administered AhR inhibitor.
According to the solution of the present invention, the IFN-γ in anti-tumor medicinal preparation combination, IDO suppression can be controlled as required
Agent and the amount of AhR inhibitor, the administration of the convenient tumor patient to different phase.
IFN-γ (i.e. gamma interferon) is a kind of water solublity dimer cytokine produced by activating T cell, has anti-
Virus, immunomodulating and antitumor properties, be a kind of common anti-tumor biological factor.Find in the applicant studies, single
Only IFN-γ can only kill the tumor cell of part, and the tumor cell not killed by IFN-γ has stem cell properties, and
Entering resting state, due to the existence of these dormancy tumor cells, tumor can recur or shift again after the several years, therefore,
IFN-γ is only administered alone and is not enough to fully erased tumor cell.The applicant is through unexpected of lot of experiments
Existing, by IFN-γ and IDO inhibitor or AhR inhibitor, combination medicine-feeding, it is possible to realize the tumor cytotoxicity to dormancy.
To sum up, the solution of the present invention has the effect that
1, the anti-tumor medicinal preparation combinatorial association IFN-γ of the application and DO inhibitor or AhR inhibitor, the most permissible
The tumor cell of differentiation is killed, and the tumor cell entering dormancy can be killed, have and thoroughly remove tumor cell
Effect.
2, the anti-tumor medicinal preparation combination of the present invention has the advantage that safety is high, have no side effect.
3, IFN-γ quantity can be reduced by this combination, thus reduce this drug-induced side reaction.
Accompanying drawing explanation
Fig. 1 shows that the CTL cell of tumour-specific can only kill part OVA-B16 tumor cell, still has fraction
OVA-B16 tumor cell survival.
Fig. 2 A is the form (light microscopic picture) after tumor cell uses the soft gel entrapment culture of 3D fibrin after different disposal again.
Fig. 2 B shows that taking equal number of tumor cell from each group plants the clone after cultivating three days in the soft gel of 3D fibrin
Number；Fig. 2 C show from each group take equal number of viable tumor cell seeding in the soft gel of 3D fibrin after three days gram
Grand number.
Fig. 3 A shows that OVA-B16 tumor cell is without the change of cell clone form under stimulating factor.Fig. 3 B shows
OVA-B16 tumor cell is at the semi-quantitative analysis relatively cloning size without cell under stimulating factor.Fig. 3 C shows OVA-
B16 tumor cell the cell cycle result under without stimulating factor.Fig. 3 D shows quantitative analysis G1/S ratio.
Fig. 4 A shows that B16 tumor cell is without the change of cell clone form under stimulating factor.Fig. 4 B shows B16
Tumor cell is relatively cloning the semi-quantitative analysis of size without cell under stimulating factor.Fig. 4 C shows that B16 tumor cell exists
Without the cell cycle result under stimulating factor.Fig. 4 D shows quantitative analysis G1/S ratio.
Fig. 5 A shows that B16 tumor cell is without the change of cell clone form under stimulating factor.Fig. 5 B shows B16
Tumor cell is cloning size relatively without cell under stimulating factor.Fig. 5 C shows that B16 tumor cell is without stimulating factor
Lower clone's number changes.Fig. 5 D shows that B16 tumor cell is without the change of cell clone form under stimulating factor.Fig. 5 E shows
B16 tumor cell is cloning size relatively without cell under stimulating factor.Fig. 5 F shows that B16 tumor cell need not stimulate
Number change is cloned under factor.
Fig. 6 A shows that associating IFN-γ and 1-MT or DMF cause the streaming figure of more tumor cell generation apoptosis
Sheet.Fig. 6 B shows the cell number ratio that apoptosis occurs in quantitative analysis Fig. 6 A.Fig. 6 C shows that the tumor after killing is thin
Born of the same parents all plant cell clone number formational situation in the soft gel of 3D fibrin.
Fig. 7 A shows that associating IFN-γ and 1-MT significantly inhibit mouse melanoma；Fig. 7 B shows associating IFN-
γ and 1-MT is obviously prolonged the life cycle suffering from melanoma mice；Fig. 7 C shows that use in conjunction IFN-γ and DMF suppress mice
Melanoma Growth；Fig. 7 D display IFN-γ and DMF therapeutic alliance murine melanoma, delay survival time of mice；Fig. 7 E shows
Melanoma to large volume, IFN-γ and 1-MT, or IFN-γ and DMF therapeutic alliance the most substantially suppress tumor growth；
Fig. 7 F shows IFN-γ and 1-MT, or IFN-γ and the bulky melanoma tumors of DMF therapeutic alliance, hence it is evident that improve mice
Life cycle；Fig. 7 G shows that associating IFN-γ and 1-MT treat the tumor caused by H22 subcutaneous injection, it is possible to substantially suppression is swollen
Tumor grows；Fig. 7 H shows IFN-γ and the growth of 1-MT use in conjunction suppression NOD-SCID murine melanoma；Fig. 7 I shows
The tumor growth that IFN-γ and 1-MT use in conjunction suppression NOD-SCID mouse subcutaneous injection MCF7 cause.
Detailed description of the invention
Various tumor cells, medicine and the laboratory animal used in following embodiment:
OVA-B16 tumor cell line, B16 mouse melanin tumor cell system (also referred to as B16 tumor cell line), H22 Mouse Liver
Cancerous cell line, MCF-7 MCF-7, all can buy from U.S. ATCC center or Chinese Typical Representative thing preservation center CCTCC.
OT-1 transgenic mice, C57BC/L mice, 4-6 week old, female Balb/c mice, or NOD-SCID mice, purchase
From Chinese Academy of Medical Sciences's Union Medical College Experimental Animal Center.
Anti-IFN-γ antibody is purchased from PepTech company；IDO inhibitor (1-MT) is purchased from SIGMA company of the U.S.；AhR suppresses
Agent (DMF), purchased from SIGMA company of the U.S..IFN-γ, the soft gel of 3D fibrin, DMEM culture medium, PBS is the most commercially available.
Embodiment 1:CTL cell can only kill part OVA-B16 tumor cell, still has fraction OVA-B16 tumor cell
Survival
1, experimental procedure
Matched group 1 (PBS+OVA-B16): with ordinary culture medium (such as DMEM culture medium)+isopyknic PBS at 96 orifice plates
Interior cultivation OVA-B16 tumor cell；
Matched group 2 (CTL+B16): B16 tumor cell and CTL cell are blended in 96 orifice plates with general with the ratio of 1:25
Logical culture medium (such as DMEM culture medium) is cultivated；
Experimental group (CTL+OVA-B16): by OVA-B16 tumor cell with CTL cell respectively with 1:1,1:10,1:25,1:
Cultivate with ordinary culture medium in 96 orifice plates after the quantitative proportion mixing of 50；
Each group tumor cell number is identical；After above-mentioned each group of tumor cell culture 4 hours, collect all cells.
From each group of cell collected on the one hand by stream measuring detection apoptosis situation (the present embodiment).On the other hand
Plant in the soft gel of 3D fibrin, cultivate observation cell clonal formation situation (see embodiment 2) in 3 days.
The preparation method of CTL cell is: separate CD8 from the spleen of OT-I transgenic mice+T cell, these tumor cells
Specific C D8+T cell becomes CTL cell (i.e. cytotoxic T lymphocyte) after AntiCD3 McAb/CD28 magnetic bead activates.Wherein OT-
I transgenic mice refers to that its TCR is the mice for OVA257-264 antigenic specificity, whole CD8 in this mice+T cell is only
Identify the OVA antigenic peptides of MHC-I quasi-molecule submission.
2, experimental result
From figure 1 it appears that tumor cell is little in matched group 1 (PBS+OVA-B16) and matched group 2 (CTL+B16)
Apoptosis；In experimental group (CTL+OVA-B16), along with the increase of CTL cell proportion, it is special to OVA-B16 tumor cell
Property lethal effect strengthen, the ratio of the OVA-B16 tumor cell of apoptosis increases to 70% from 15%.But, still have part (about
30%) OVA-B16 tumor cell can not be killed by CTL cell.
Embodiment 2: the tumor cell can not killed by CTL cell has tumor stem cell characteristic.
1, experimental procedure
By 4 hours (step is as described in example 1 above) of above-mentioned each group of tumor cell culture, and after collecting all cells, real
Impose lower operation: 1) take equal number of tumor cell from each group and plant in the soft gel of 3D fibrin (1mg/mL fibrin)
In；2) tumor cell number survived in the CTL+OVA-B16 group (being called for short 1:50 group) with OVA-B16:CTL as 1:50 is as standard,
Take equal number of viable tumor cell seeding in the soft gel of 3D fibrin；3) observation of cell growing state after 3 days.
This soft gel of 3D fibrin known is unfavorable for the growth of normal tumor cell, but is suitable for screening tumor regrowth
Cell (TRC), i.e. has the tumor cell of stem cell properties, it is possible to be referred to as tumor stem cell.Own in tumor cell normally
Also the tumor stem cell of constant ratio is contained.Only tumor stem cell can form cell clone in the soft gel of 3D fibrin.
2, experimental result
Fig. 2 A is each group of tumor cell form.
Fig. 2 B is to take equal number of tumor cell from each group to plant and cultivate after three days in the soft gel of 3D fibrin
Experimental data, it can be seen that the plastidogenetic clone's number of each group is close.This be due to all experimental grouies and cellular control unit from
Same type of tumor cell, itself tumor stem cell containing constant ratio in these tumor cells, when through CTL cell killing
After each group tumor cell all plant in the above-mentioned soft gel of 3D fibrin with identical cell number with matched group tumor cell
Time, due to the tumor stem cell containing same ratio, the colony counts grown out in these groups is close to identical.
Fig. 2 C is for taking equal number of viable tumor cell seeding in the soft gel of 3D fibrin from each group, after 3 days
Experimental data, can see in 1:50 group, its Clone formation number showed increased, compared to other groups, has and statistically has
Significant difference.This be due to the tumor cell number of survival in 1:50 group (include dormancy tumor cell and normal the most not by
The tumor cell killed) on the basis of plant, the tumor cell plantation that the most each group only takes survival, the viable tumor cell of plantation
Number is identical with viable tumor cell number in 1:50 group, owing to the CTL added in 1:50 group is most, at these tumor cells survived
The ratio of middle dormancy tumor stem cell is the highest, and remaining each group few due to the CTL added, and corresponding dormancy tumor stem cell ratio is low
And the ratio of the tumor cell not killed is high, therefore in 1:50 group, its Clone formation number is significantly more than other groups.Simultaneously
Also the tumor cell that explanation can not be killed by CTL cell has tumor stem cell characteristic.
The supernatant that embodiment 3:CTL cell and OVA-B16 tumor cell obtain after hatching altogether can cause OVA-B16 tumor
Cell enters dormancy.
1. experimental procedure:
Matched group (PBS): normal OVA-B16 tumor cell is planted in the soft gel of 3D fibrin, uses Nostoc commune Vanch
Base (such as DMEM culture medium)+isopyknic PBS cultivates 3-7 days, and observation of cell growing state also carries out the cell cycle；
Experimental group 1 (INF-γ): OVA-B16 tumor cell is planted in the soft gel of 3D fibrin, uses Nostoc commune Vanch
After base is cultivated 2 days into, this ordinary culture medium is changed the culture medium (i.e. conditioned medium 1) of ordinary culture medium+equal-volume supernatant,
This supernatant is the supernatant obtained after CTL cell is hatched altogether with OVA-B16 tumor cell, containing INF-γ in this supernatant,
Continuation cultivation OVA-B16 tumor cell is after 3-5 days, and observation of cell growing state also carries out the cell cycle.
Experimental group 2 (anti-INF-γ): by OVA-B16 cell seeding in the soft gel of 3D fibrin, use ordinary culture medium
After cultivating 2 days into, this ordinary culture medium is changed culture medium (the i.e. condition of ordinary culture medium+equal-volume supernatant+anti-INF-γ
Culture medium 2), this supernatant is the supernatant that CTL cell is hatched altogether with OVA-B16 cell, containing INF-γ in this supernatant, continues
Continuous OVA-B16 tumor cell of cultivating is after 3-5 days, then observation of cell growing state carry out the cell cycle.
The supernatant that CTL cell and OVA-B16 tumor cell obtain after hatching altogether such as can be from embodiment 1 experimentation
Middle acquisition.
The cell cycle is carried out according to test kit description.
2. experimental result:
Comparing matched group, conditioned medium 1 stimulates the tumor cell of OVA-B16 after 3 days, and cell clone size substantially subtracts
Little, continue to cultivate 2 days, cell clone size keeps constant；But, use ordinary culture medium if changed into again by conditioned medium 1
Rear cultivation 2 days, cell clone the most substantially becomes greatly that (the string data of the 7th day test number for changing ordinary culture medium in the middle of Fig. 3 A
According to), implied condition culture medium 1 induces OVA-B16 cell to enter dormancy.If being simultaneously introduced anti-INF-in conditioned medium 1
γ i.e. conditioned medium 2, then can the dormant cells that causes of reverse transfer condition culture medium 1, the IFN-γ in implied condition culture medium 1 is
Cause the factor (see Fig. 3 A, Fig. 3 B) of dormant cells.The result of the cell cycle is consistent with the above results, i.e. conditioned medium
Causing cell block in the G0/G1 phase, anti-INF-gamma antibodies then can reverse this process (see Fig. 3 C, Fig. 3 D).
Embodiment 4:IFN-γ causes B16 tumor cell to enter dormancy.
1. experimental procedure:
B16 tumor cell is carried out following operation:
Matched group (PBS): 3000 normal B16 tumor cells are planted in the 3D fibrin being positioned on 24 orifice plates soft solidifying
In glue (single is 250 μ L containing gel volume in gel pore, and the ordinary culture medium that gel adds above is 1mL), use Nostoc commune Vanch
Base+PBS (the least only 1 microlitre of its volume added) cultivates 3-8 days；Observation of cell growing state also carries out cell cycle survey
Fixed；
Experimental group (IFN-γ): B16 tumor cell is planted in the soft gel of 3D fibrin, cultivates 2 with ordinary culture medium
After it, this ordinary culture medium changing the culture medium of ordinary culture medium+IFN-γ into, the concentration of IFN-γ is respectively 50ng/mL,
100ng/mL, stimulates B16 tumor cell after 3-5 days, and observation of cell growing state also carries out the cell cycle.
Wherein IFN-γ is >=5U/ng, and the cell cycle is to carry out according to test kit description.
2. experimental result:
Extending with incubation time, in matched group, B16 tumor cell clone becomes larger；In experimental group, to B16 tumor cell
With the IFN-γ of 100ng/mL stimulate latter 3 days compared with stimulating latter 5 days, cell clone size remains unchanged (see Fig. 4 A, Fig. 4 B).
If removed latter 3 days by stimulating factor IFN-γ, cell clone size starts again to significantly increase (the middle string of Fig. 4 A and the right
The string data of the 8th day are the experimental data after changing ordinary culture medium into).
Consistent with this result, the cell cycle result shows, IFN-γ stimulates cell cycle arrest in G0/G1
Phase, its G1/S phase ratio significantly raised compared to matched group (see Fig. 4 C, Fig. 4 D).
Embodiment 5:IDO inhibitor reverses the Tumor dormancy that IFN-γ causes.
1. experimental procedure:
B16 tumor cell is carried out following operation:
Matched group 1 (PBS): the 3D fibrin planting 3000 normal B16 tumor cells in being positioned on 24 orifice plates is soft
In gel (single is 250 μ L containing gel volume in gel pore, and the ordinary culture medium that gel adds above is 1mL), with common training
Support base+PBS (the least only 1 microlitre of its volume added) to cultivate 3-5 days；
Matched group 2 (IFN-γ): B16 tumor cell is planted in the soft gel of 3D fibrin, trains with ordinary culture medium
After supporting 2 days, ordinary culture medium adds IFN-γ (100ng/mL) and processes 3-5 days；
Experimental group: planted by B16 tumor cell in the soft gel of 3D fibrin, cultivates 24 hours with ordinary culture medium
After, ordinary culture medium adds IFN-γ (100ng/mL) and 1-MT (0.2mM) processes cell 3-5 days, or at Nostoc commune Vanch
Base adds IFN-γ (100ng/mL) and DMF (20 μMs) processes cell 3-5 days；
Detecting each group of cell clone size and Clone formation number, wherein IFN-γ is >=5U/ng.
2. experimental result:
B16 tumor cell is after IFN-γ and 1-MT combined stimulation, and it is bright that its clone's size compares IFN-γ individual processing group
Aobvious reduction；And extending with stimulation time, its clone's size becomes more and more less (see Fig. 5 A, Fig. 5 B).Counting colony counts is sent out
Existing, after use in conjunction IFN-γ and 1-MT, clone's number that B16 tumor cell is formed significantly reduces, and comparing matched group has significance difference
Different (see Fig. 5 C).
Similar with this result, B16 tumor cell is after IFN-γ and DMF combined stimulation, and its clone's size compares IFN-γ
Individual processing group is obviously reduced；And extending with stimulation time, its clone's size becomes more and more less (see Fig. 5 D, Fig. 5 E).Gram
Grand number count results shows, after use in conjunction IFN-γ and DMF stimulate, clone's number that B16 tumor cell is formed significantly reduces,
There were significant differences (see Fig. 5 F) to compare matched group.
Embodiment 6: use in conjunction CTL cell and IDO inhibitor, or use in conjunction CTL cell and AhR inhibitor can kill
OVA-B16 tumor cell, particularly can kill the tumor cell of the OVA-B16 of dormancy.
1. experimental procedure:
Matched group (PBS): 5000 OVA-B16 tumor cells are planted in 96 orifice plates (single pore volume is 100 micro-
Rise), only cultivate with ordinary culture medium and add equal-volume PBS cultivation 3-5 days；
Matched group 2 (1-MT): plant in 96 orifice plates by 5000 OVA-B16 tumor cells is little with ordinary culture medium 18
Shi Hou, adds 1-MT (0.2mM or 0.5mM) process OVA-B16 tumor thin in ordinary culture medium (such as DMEM culture medium)
Born of the same parents 3-5 days；
Matched group 3 (DMF): plant in 96 orifice plates by 5000 OVA-B16 tumor cells, with ordinary culture medium 18 hours
After, ordinary culture medium adds DMF (20 μMs or 50 μMs) and processes OVA-B16 tumor cell 3-5 days；
Experimental group: 5000 OVA-B16 tumor cells are planted in 96 orifice plates, after ordinary culture medium 18 hours, so
After in ordinary culture medium, add from OT-I mouse spleen isolated CTL cell (wherein CTL cell and OVA-B16 tumor
The quantitative proportion of cell is 30-50:1) and 1-MT (0.2mM or 0.5mM), or in ordinary culture medium, add described CTL
Cell and DMF (20 μMs or 50 μMs), stimulate OVA-B16 tumor cell after 24 hours, collect all tumor cells: on the one hand examine
Survey apoptosis of tumor cells situation；On the other hand equal number of tumor cell is planted in the soft gel of 3D fibrin, cultivate 3
It observes cell clonal formation situation.
2. experimental result:
It is used alone CTL cell and causes about 38%OVA-B16 apoptosis of tumor cells, be used alone 1-MT or DMF process
Cell does not cause significant apoptosis (see Fig. 6 A, Fig. 6 B).But, use in conjunction CTL cell and 1-MT then cause about
60% apoptosis, compares and is used alone CTL cell process group, and difference has significance (see Fig. 6 A, Fig. 6 B).Tie with this
Fruit seemingly, is used in combination CTL cell and DMF causes more OVA-B16 tumor cell generation apoptosis, and is used alone CTL cell
Process group is compared, and its apoptotic cell number adds about one times, and along with DMF dosage increases, its apoptotic cell number is bright
Show and increase (see Fig. 6 A, Fig. 6 B).
The equal number of OVA-B16 cell seeding processed through different factors is found in the soft gel of 3D fibrin,
Being used alone CTL groups of cells or be used alone 1-MT process group, its cell clonal formation number nothing compared with matched group is notable
Difference；But, use in conjunction CTL is compared with 1-MT and is used alone the process of CTL cell, then cell clone number significantly reduces (220
Individual ratio 360) (see Fig. 6 C).
Being used alone DMF process, in the case of high dose, its cell clonal formation number relatively matched group significantly reduces；And
CTL cell and DMF Combined Treatment the most less cell clonal formation number (see Fig. 6 C).
Test result indicate that: use in conjunction CTL cell and IDO inhibitor, or use in conjunction CTL cell and AhR inhibitor
OVA-B16 tumor cell can be killed, particularly can kill the tumor cell of the OVA-B16 of dormancy.
Embodiment 7: use in conjunction IFN-γ and IDO inhibitor, or IFN-γ and AhR inhibitor can suppress tumor-bearing mice swell
Tumor grows, and extends life cycle.
1, use in conjunction IFN-γ and IDO inhibitor (seeing Fig. 7 A and Fig. 7 B):
1) experimental procedure
Structure tumor-bearing mice: 4-6 week C57BL/C mice 40, is randomly divided into 4 groups, and Mouse Weight is 20g；Every mouse
Subcutaneous vaccination 3 × 104B16 mouse melanin tumor cell, it is observed that tumor generates after 10 days；Inoculation melanoma cell the 3rd
It starts different modes following to tumor-bearing mice respectively and is administered:
Matched group 1 (Con), to tumor-bearing mice only tail vein injection PBS；
Matched group 2 (IFN-γ): IFN-γ individual processing tumor-bearing mice, every rat tail intravenous injection 1 × 105U's
IFN-γ, injection in every three days once, is injected 3 times altogether；
Matched group 3 (1-MT): 1-MT individual processing tumor-bearing mice, is dissolved in the drink of mice by 1-MT according to the dosage of 5mg/mL
With water, every mouse about drinks 3-4mL drinking water, successive administration 10 days every day；
Experimental group 1 (IFN-γ/1-MT): tumor-bearing mice is given respectively IFN-γ and 1-MT (i.e. use in conjunction IFN-γ
And 1-MT), medication is the same.
Melanoma cell inoculation after the 13rd day, start to measure tumor size, and observe survival time of mice.
2) experimental result
IFN-γ or the independent medication of 1-MT cause mice tumors grew slow, and use in conjunction IFN-γ and 1-MT then lead
Causing obvious Tumor growth inhibition, after inoculation melanoma cell, after the 23rd day, mouse tumor volume reduces by 70%, compares comparison
Group, difference has significant (see Fig. 7 A).Observation survival time of mice finds, is used alone IFN-γ or 1-MT extends little
Mus life cycle, and use in conjunction IFN-γ and 1-MT are to the prolongation effect of survival time of mice the most substantially (see Fig. 7 B).
2, use in conjunction IFN-γ and AhR inhibitor (seeing Fig. 7 C and Fig. 7 D)
1) experimental procedure
Build tumor-bearing mice method with aforementioned item 1.Inoculate melanoma cell after 10 days it is observed that tumor generates；Inoculation
Melanoma cell starts different modes following to tumor-bearing mice respectively on the 3rd day and is administered:
Matched group 1 (Con), to tumor-bearing mice only tail vein injection PBS；
Matched group 2 (IFN-γ): IFN-γ individual processing tumor-bearing mice, every rat tail intravenous injection 1 × 105U's
IFN-γ, injection in every three days once, is injected 3 times altogether；
Matched group 3 (DMF): DMF individual processing tumor-bearing mice, DMF consumption is 5mg/kg Mouse Weight every day, administering mode
For gavage, it is administered 10 days；
Experimental group 1 (IFN-γ/DMF): mice gives IFN-γ and DMF (i.e. use in conjunction IFN-γ and DMF) respectively, gives
Prescription method is the same.
Melanoma cell is inoculated the 13rd day, starts to measure tumor size, and observes survival time of mice.
2) experimental result
IFN-γ or the independent medication of DMF cause mice tumors grew slow, and use in conjunction IFN-γ and DMF then cause
Significantly Tumor growth inhibition, after inoculation melanoma cell, after the 23rd day, mouse tumor volume reduces by 70%, compares matched group,
Difference has significant (see Fig. 7 C).Observation survival time of mice finds, is used alone IFN-γ or DMF extends mice life
Deposit the phase, and use in conjunction IFN-γ and DMF are the most obvious (see Fig. 7 D) to the prolongation effect of survival time of mice.
3, IDO or the AhR inhibitor Experiment on therapy (seeing Fig. 7 E, Fig. 7 F and Fig. 7 G) to bulky tumors
1) experimental procedure
The structure of tumor-bearing mice: by 1 × 105B16 mouse melanin tumor cell is subcutaneous, cultivates 10 days, it was observed that tumor is long
To 7 millimeters × 7 millimeters (the most bulky melanoma tumors), then within the 11st day, start different modes following to tumor-bearing mice respectively
It is administered:
Matched group (Con), to tumor-bearing mice only tail vein injection PBS；
Experimental group 1 (DMF/IFN-γ): mice gives IFN-γ and DMF (i.e. use in conjunction IFN-γ and DMF) respectively；
DMF administering mode is: every three days intratumor injection administration DMF 5mg/kg Mouse Weights, totally 3 times；IFN-γ administering mode is: every
Within three days, intratumor injection is administered 1 × 105The IFN-γ of U, totally 3 times；
Experimental group 2 (1-MT/IFN-γ): mice gives IFN-γ and 1-MT (i.e. use in conjunction IFN-γ and 1-respectively
MT), 1-MT administering mode is: intratumor injection is administered (250ug/ Mouse Weight is given once, 3 times altogether for every three days)；IFN-γ
Administering mode is: within every three days, intratumor injection is administered 1 × 105The IFN-γ of U, altogether injection 3 times；
Melanoma cell inoculation after the 10th day, start to measure tumor size, and observe survival time of mice.
2) experimental result
Compared to matched group, IFN-γ and 1-MT, or IFN-γ and DMF therapeutic alliance substantially suppress melanoma tumor raw
Long (see Fig. 7 E), and significantly extend survival time of mice (see Fig. 7 F).
Consistent with this result, by 1 × 105It is little that H22 murine hepatocarcinoma cell is inoculated into C57BL/C mice subcutaneous structure lotus tumor
Mus, after 13 days, it was observed that tumor grows to 7 millimeters × 7 millimeters, start simultaneously at measurement tumor size, and start at the 14th day to be administered,
Ibid, IFN-γ and 1-MT therapeutic alliance also significantly inhibit the large volume (7 millimeters that H22 murine hepatocarcinoma cell causes to administering mode
× 7 millimeters) tumor growth (see Fig. 7 G), IFN-γ is used in combination and also can obtain similar effect (not shown) with DMF.These knots
Fruit explanation IFN-γ and 1-MT, or IFN-γ and DMF therapeutic alliance are not only effective to small size tumor, and to bulky tumors
Treat also effect notable.
4, IDO or the AhR inhibitor Experiment on therapy (seeing Fig. 7 H and Fig. 7 I) to NOD-SCID mouse tumor
1) experimental procedure
Build lotus tumor NOD-SCID mice: for confirming that whether IDO or AhR inhibitor is to pass through to the inhibitory action of tumor
The immunoreation of regulation whole body, 6 weeks NOD-SCID mices 20, every mouse hypodermic inoculation 2 × 105B16 murine melanoma
Cell, after 8 days, it was observed that tumor is formed, and lotus tumor NOD-SCID mice is randomly divided into three groups, in inoculated tumour cell the 3rd day
Start to be administered with following different modes:
Matched group (Con): lotus tumor NOD-SCID mice is carried out tail vein injection PBS；
Experimental group (1-MT/IFN-γ): lotus tumor NOD-SCID mice is carried out tail vein injection IFN-γ, injection in every three days
Once, injection three times altogether, wherein IFN-γ is 1 × 105U/ mice/time, it is administered simultaneously 1-MT, is dissolved according to the dosage of 5mg/mL
The drinking water of mice, every mouse is about drunk 3-4mL drinking water, be administered 10 days every day.
Melanoma cell inoculation after the 8th day, start to measure tumor size.
2) experimental result
During nearly three weeks of observation, use in conjunction IFN-γ and 1-MT substantially suppress tumor growth, compare matched group P
< 0.001 (see Fig. 7 H).We are by the 2 × 10 of people6MCF-7 human breast cancer cell is expelled to NOD-SCID mice subcutaneous structure lotus
Tumor mice, observed that tumor is formed after 6 days, started to measure tumor size at the 6th day, inoculated tumour cell within the 3rd day, start to
Medicine, administering mode ibid, also obtains similar results, shows as IFN-γ and 1-MT drug combination significantly inhibits MCF-7 people's mammary gland
The tumor growth (see Fig. 7 I) that cancerous cell causes.Illustrate that the growth of IDO or AhR inhibitors to inhibitor tumor is not by regulation whole body
Immunoreation is carried out.
Claims (8)
1. anti-tumor medicinal preparation combination, it is characterised in that include IFN-γ and IDO inhibitor, or IFN-γ and AhR
Inhibitor.
Anti-tumor medicinal preparation the most according to claim 1 combines, it is characterised in that described tumor includes ovarian cancer, breast
Adenocarcinoma, pulmonary carcinoma, gastric cancer, colon cancer, hepatocarcinoma, cancer of pancreas or carcinoma of prostate.
Anti-tumor medicinal preparation the most according to claim 1 combines, it is characterised in that described IDO inhibitor includes 1-MT.
Anti-tumor medicinal preparation the most according to claim 1 combines, it is characterised in that described AhR inhibitor includes DMF.
Anti-tumor medicinal preparation the most according to claim 1 combines, it is characterised in that described anti-tumor medicinal preparation combines
In IFN-γ, IDO inhibitor and AhR inhibitor be unit preparation.
Anti-tumor medicinal preparation the most according to claim 5 combines, and described IFN-γ unit formulation includes 1 × 107～4
×107The IFN-γ of U.
Anti-tumor medicinal preparation the most according to claim 5 combines, and described IDO inhibitor unit formulation includes 4～8g
IDO inhibitor.
Anti-tumor medicinal preparation the most according to claim 5 combines, and described AhR inhibitor includes that the AhR of 27-50mg presses down
Preparation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109966275A (en) * | 2017-12-11 | 2019-07-05 | 北京芳草园生物科技有限公司 | Quinoid chalcone compound application in preparation of anti-tumor drugs |
| WO2022016844A1 (en) * | 2019-07-22 | 2022-01-27 | 厦门特宝生物工程股份有限公司 | Interferon-based cancer treatment method and pharmaceutical composition |
| CN114010791A (en) * | 2021-11-15 | 2022-02-08 | 济南市中心医院 | Application of MyD88-IFN gamma R1 dimer as target in preparation of medicine for preventing colon cancer |
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| WO2008068621A2 (en) * | 2006-12-05 | 2008-06-12 | Molmed Spa | Combination product with ido inhibitor and tumor targeted ifn-gamma |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008068621A2 (en) * | 2006-12-05 | 2008-06-12 | Molmed Spa | Combination product with ido inhibitor and tumor targeted ifn-gamma |
Non-Patent Citations (2)
| Title |
|---|
| TING WEN CHUNG ETAL: "Induction of Indoleamine 2,3-dioxygenase (IDO) Enzymatic Activity Contributes to Interferon-Gamma Induced Apoptosis and Death Receptor 5 Expression in Human Non-small Cell Lung Cancer Cells", 《ASIAN PACIFIC JOURNAL OF CANCER PREVENTION》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109966275A (en) * | 2017-12-11 | 2019-07-05 | 北京芳草园生物科技有限公司 | Quinoid chalcone compound application in preparation of anti-tumor drugs |
| CN109966275B (en) * | 2017-12-11 | 2021-11-30 | 谷冲医药(北京)股份有限公司 | Application of quinoid chalcone compound in preparation of antitumor drugs |
| WO2022016844A1 (en) * | 2019-07-22 | 2022-01-27 | 厦门特宝生物工程股份有限公司 | Interferon-based cancer treatment method and pharmaceutical composition |
| CN114010791A (en) * | 2021-11-15 | 2022-02-08 | 济南市中心医院 | Application of MyD88-IFN gamma R1 dimer as target in preparation of medicine for preventing colon cancer |
| CN114010791B (en) * | 2021-11-15 | 2023-11-24 | 济南市中心医院 | Application of MyD88-IFN gamma R1 dimer as target in preparation of medicine for preventing colon cancer |
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