CN106110322A - A kind of pharmaceutical composition and the application in preparation treatment cancer drug thereof - Google Patents
A kind of pharmaceutical composition and the application in preparation treatment cancer drug thereof Download PDFInfo
- Publication number
- CN106110322A CN106110322A CN201610615271.7A CN201610615271A CN106110322A CN 106110322 A CN106110322 A CN 106110322A CN 201610615271 A CN201610615271 A CN 201610615271A CN 106110322 A CN106110322 A CN 106110322A
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- Prior art keywords
- cancer
- pharmaceutical composition
- monoclonal antibody
- bacillus bifidus
- tumor
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
Abstract
The invention discloses a kind of pharmaceutical composition and the application in preparation treatment cancer drug thereof, belong to biological pharmacy technical field.Pharmaceutical composition provided by the present invention is made up of PD 1 monoclonal antibody and bacillus bifidus.Wherein, the quality of PD 1 monoclonal antibody and the volume combination matching of bacillus bifidus bacterium solution are 1～2mg:1～2ml.The usage amount of PD 1 monoclonal antibody is 2mg/kg, and the viable count of bacillus bifidus is 1 107cfu/mL.Meanwhile, the pharmaceutical composition that the present invention provides can be applied in the medicine of preparation treatment cancer.Pharmaceutical composition provided by the present invention has good tumor inhibition effect, it is possible to the growth of the tumors such as effective suppression melanoma.
Description
Technical field
The present invention relates to a kind of pharmaceutical composition and the application in preparation treatment cancer drug thereof, belong to bio-pharmaceuticals skill
Art field.
Background technology
Programmed death receptor 1 (Programmed Death-1；PD-1), it is a kind of important immunosuppression molecule, for
CD28 superfamily member, it is initially that the mouse Tcell hybridoma 2B4.11 from apoptosis clones out.PD-1 is mainly activating
T cell and B cell in express, there is the function of suppression cell-stimulating, this is a kind of normal homeostasis in immune system,
Because excessive T/B cell-stimulating can cause autoimmune disease, so PD-1 is equivalent to one guardrail in human body.
In tumor microenvironment, part PD-L1 (apoptosis-ligand 1 or the table of tumor cell meeting high expressed PD-1
Face antigen differentiation bunch 274 or B7 autoploids).The PD-L1 expressed on tumor cell and the PD-1 on T cell surface interacts, and leads
Causing PD-1 path sustained activation in tumor microenvironment, T cell function is suppressed, it is impossible to killing tumor cell.PD-1 monoclonal anti
Body can by blocking this path, part recover T cell function, and then utilize activate T cell and corresponding cell because of
Sub-killing tumor cell, thus it becomes the critical treatment means of immunotherapy for cancer.
Research shows, bacillus bifidus also has important effect at anti-tumor aspect.Bacillus bifidus is from breast milk nutrition youngster
Feces in the Gram-positive bacillus of isolated a kind of anaerobism, it can suppress intestinal by adjusting normal intestinal flora
Many saprophytic bacteria growths, thus reduce some carcinogen and produce, bacillus bifidus has activation macrophage in body or LAK cell
Phagocytic activity, and produce a certain amount of cytokine such as TNF a INF V etc. and can directly kill tumor cell.Suppression tumor
Interior vascularization, destroys tumor tissues blood capillary, ultimately results in hemorrhage, downright bad.Due to PD-1 monoclonal antibody and bacillus bifidus,
One belongs to biomacromolecule material, and one belongs to probiotic bacteria, often both is used separately in prior art, a kind of directly work
For drug use, another kind then adds health food as prebiotics or milk product is prepared in fermentation, the most will not in prior art
The two mixed report.
Summary of the invention
For solving the problems referred to above, the invention provides a kind of pharmaceutical composition and this pharmaceutical composition in preparation treatment cancer
Purposes in disease drug, the technical scheme taked is as follows:
It is an object of the invention to provide a kind of pharmaceutical composition, this medicine is by PD-1 monoclonal antibody and bacillus bifidus bacterium
Two kinds of one-tenth of liquid are grouped into.
Preferably, the quality (mg) of described PD-1 monoclonal antibody and volume (ml) combination matching of bacillus bifidus be 1～
2:1～2.
It is highly preferred that volume (ml) combination matching of the quality (mg) of described PD-1 monoclonal antibody and bacillus bifidus is 1:
1。
It is highly preferred that the use content of described PD-1 monoclonal antibody is 2mg/kg；The viable count of described bacillus bifidus is 1
×107cfu/mL。
The application in preparation treatment cancer drug of the pharmaceutical composition described in any of the above.
Preferably, described cancer is pulmonary carcinoma, gastric cancer, hepatocarcinoma, colorectal carcinoma, melanoma, nephroncus, ovarian cancer, prostatitis
Adenocarcinoma, bladder cancer, breast carcinoma, the esophageal carcinoma, colorectal cancer, nasopharyngeal carcinoma, the cerebral tumor, cervical cancer, leukemia, osteocarcinoma, lymphatic cancer, pancreas
Cancer.
Preferably, described cancer is melanoma.
The beneficial effect that the present invention obtains:
PD-1 monoclonal antibody is a kind of immunosuppression molecule in human or animal, and bacillus bifidus is a kind of for milk product
Probiotic bacteria in processing.Creative both being used in combination of inventor, by experimental verification, both discoveries are used in combination can
The growth of highly effective suppression tumor, the two there is synergism, it is possible to significantly improve PD-1 mab treatment
Effect of cancer.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention should not be limited by the examples.
Following example material therefor, reagent, method and instrument, without specified otherwise, be this area conventional material, examination
Agent, method and instrument, those skilled in the art all can be obtained by commercial channel.
Embodiment 1
PD-1 monoclonal antibody and the bacillus bifidus therapeutic alliance inhibitory action to mice melanotic tumor
(1) foundation of murine melanoma model uses subcutaneous inhibition, it is simple to observe tumor growth situation.Aseptic behaviour
Making the lower seroperitoneum extracting seroperitoneum type B16 melanoma Mus, Trypan Blue checks cancerous cell survival rate, and survival rate is big
Seroperitoneum oncocyte serum-free medium in 95% is adjusted to 1 × 107/mL.Taking normal 6 week old nude mice 25, every old
Mus body weight is about 20g, subcutaneous injection 0.2mL cell suspension at Mus Mus Xi on the right side of every nude mice, 10d in inoculate out grow 2～
During 3mm tumor, murine melanoma modelling success.
(2) it is grouped and processes
After modeling, mice is randomly divided into 5 groups, and often group 5, bacillus bifidus and PD-1 antibody PBS dissolves, and includes PBS respectively
Matched group (lumbar injection sterilizing PBS1mL/d), PD-1 monoclonal antibody process group (lumbar injection 1mL 2mg/kg/d), PD-1 monoclonal antibody and
Bacillus bifidus Combined Treatment group (PD-1 monoclonal antibody lumbar injection 1mL 2mg/kg/d and bacillus bifidus 1 × 107Cfu/mL), bifid bar
Bacterium process group (1mL bacillus bifidus 1 × 107Cfu/mL), CTX (chemotherapeutic cyclophosphamide) (lumbar injection CTX 15mg/kg/d),
Start to be administered with next day after inoculation oncocyte, successive administration 12d.
(3) research to murine melanoma inhibitory action
Drug withdrawal next day execution mice of weighing, dissect and peel off tumor mass, claim tumor weight, by average tumor re-computation tumour inhibiting rate.Press down
Ratio of outflow=[(matched group gross tumor volume-experimental group gross tumor volume)/matched group gross tumor volume] × 100%.
(4) experimental result
Result shows, process group is compared with PBS control group, and the former substantially inhibits the growth of mice B16 melanoma,
The tumour inhibiting rate of PD-1 monoclonal antibody process group, PD-1 monoclonal antibody and bacillus bifidus Combined Treatment group and bacillus bifidus process group is respectively
35.83%, 31.33%, 55.31%.Wherein, PD-1 monoclonal antibody is compared with other two groups with bacillus bifidus Combined Treatment group, has aobvious
Write difference (P < 0.05). illustrating, PD-1 monoclonal antibody and bacillus bifidus are used in combination suppression melanoma effect and are higher than other each group
(being shown in Table 1).
Each group of inhibition to murine melanoma of table 1
Embodiment 2
Impact that blood vessels in tumors is generated by PD-1 monoclonal antibody and bacillus bifidus therapeutic alliance and cell cycle and
The change of apoptosis.
(1) foundation of murine melanoma model uses subcutaneous inhibition, it is simple to observe tumor growth situation.Aseptic behaviour
Making the lower seroperitoneum extracting seroperitoneum type B16 melanoma Mus, Trypan Blue checks cancerous cell survival rate, and survival rate is big
Seroperitoneum oncocyte serum-free medium in 95% is adjusted to 1 × 107/mL.Taking normal 6 week old nude mice 25, every old
Mus body weight is about 20g, subcutaneous injection 0.2mL cell suspension at Mus Mus Xi on the right side of every nude mice, 10d in inoculate out grow 2～
During 3mm tumor, murine melanoma modelling success.
(2) it is grouped and processes
After modeling, mice is randomly divided into 5 groups, and often group 5, bacillus bifidus and PD-1 antibody PBS dissolves, PBS control group
(lumbar injection sterilizing PBS1mL/d), PD-1 monoclonal antibody process group (lumbar injection 1mL 2mg/kg/d), PD-1 monoclonal antibody and bifid bar
Bacterium Combined Treatment group (PD-1 monoclonal antibody lumbar injection 1mL 2mg/kg/d and bacillus bifidus 1 × 107Cfu/mL), bacillus bifidus processes
Group (1mL bacillus bifidus 1 × 107Cfu/mL), CTX (chemotherapeutic cyclophosphamide) (lumbar injection CTX 15mg/kg/d), with inoculation
After oncocyte, next day starts to be administered, successive administration 12d.
(3) VIII-RAg SABC and flow cytomery murine melanoma vascularization, cell cycle and apoptosis
Rate
Formation by immunohistochemical kit detection murine melanoma blood vessel: conventional dewaxing is to water；Mass fraction is 3%
H2O2Hatch to eliminate endogenous catalase activity；Priority citrate buffer, mass fraction are the pancreas of 0.1%
Protease repairs antigen；Specimen is after confining liquid effect, and the one of dropping proper proportion dilution resists (VIII-RAg Anti-TNF-α successively
Body), the biotin labeled two anti-and strepto-avidins of horseradish peroxidase-labeled；DAB develops the color, and haematoxylin redyes core；Ladder
Degree ethanol dehydration, dimethylbenzene is transparent, neutral gum mounting.Light Microscopic observation vascular endothelial cell staining conditions.
Cell cycle and apoptosis rate detection: fresh tumor is made single-cell suspension liquid 1 × 107/ L, is centrifuged and abandons supernatant,
Add DNAPrepL RP 210 μ l, DNA-Prep Stain 2.2ml, mixing, lucifuge, dyeing, room temperature 30min.Thin by streaming
Born of the same parents' instrument carries out detecting (acquisition of Cellquese software), it is thus achieved that DNA rectangular histogram, according to measured DNA histogram, uses cell
Cycle analysis software modfit carries out the distribution of cell cycle each phase and the analysis of apoptosis rate.
(4) experimental result
Result shows, process group is compared with PBS control group, and process group substantially inhibits the blood vessel of mice B16 melanoma
Formed, PD-1 monoclonal antibody process group, PD-1 monoclonal antibody and bacillus bifidus Combined Treatment group and the most each the regarding of bacillus bifidus process group
Wild microvessel count is respectively 61,42,77.Utilize SPSS10 software analysis, PD-1 monoclonal antibody and the micro-blood of bacillus bifidus drug combination
Pipe number is less than other each group, and reaches significance of difference P < 0.05.
The result that cell cycle and apoptosis rate are analyzed shows, process group, compared with PBS control group, can make cell cycle hinder
Stagnant in the S phase, suppression Tumour DNA synthesis, and then suppression tumor cell proliferation.PD-1 monoclonal antibody and the resistance of bacillus bifidus drug combination group
Stagnant effect is higher than other each group, wherein matched group, PD-1 monoclonal antibody process group, bacillus bifidus process group, PD-1 monoclonal antibody and bifid bar
Bacterium Combined Treatment group and CTX process group S phase cells ratio be respectively 13.23%, 17.81%, 18.74%, 21.66%,
15.37%.Apoptosis of tumor cells rate is followed successively by PD-1 monoclonal antibody and bacillus bifidus Combined Treatment group from big to small > process of PD-1 monoclonal antibody
Group > bacillus bifidus process group > CTX process group > matched group.Experimental result is shown in Table 2 and table 3.
The medicine impact on the B12 melanoma cell cycle respectively organized by table 2
The medicine impact on B12 melanoma cell apoptosis rate respectively organized by table 3
| Use medicine | Apoptosis of tumor cells rate |
| PBS (compares) | (0.77 ± 0,031) % |
| PD-1 monoclonal antibody | (12.87 ± 2.37) % |
| Bacillus bifidus | (1.87 ± 0.04) % |
| PD-1 monoclonal antibody & bacillus bifidus | (22.76 ± 1.57) % |
| CTX | (1.53+0.38) % |
Embodiment 3
PD-1 monoclonal antibody and the optimal proportion of bacillus bifidus therapeutic alliance mice melanotic tumor.
(1) foundation of murine melanoma model uses subcutaneous inhibition, it is simple to observe tumor growth situation.Aseptic behaviour
Making the lower seroperitoneum extracting seroperitoneum type B16 melanoma Mus, Trypan Blue checks cancerous cell survival rate, and survival rate is big
Seroperitoneum oncocyte serum-free medium in 95% is adjusted to 1 × 107/mL.Taking normal 6 week old nude mice 25, every old
Mus body weight is about 20g, subcutaneous injection 0.2mL cell suspension at Mus Mus Xi on the right side of every nude mice, 10d in inoculate out grow 2～
During 3mm tumor, murine melanoma modelling success.
(2) it is grouped and processes
After modeling, mice is randomly divided into 9 groups, and often group 5, is respectively designated as 1～9 group, often organizes the use quality of PD-1 antibody
(mg) volume (ml) ratio with bacillus bifidus is respectively as follows: 1:1,1:2,1:3,1:4,1:5,5:1,4:1,3:1,2:1, wherein PD-1
The dosage of monoclonal antibody lumbar injection is 2mg/kg/d, and the concentration of bacillus bifidus is 1 × 107cfu/mL.After inoculation oncocyte
Start next day to be administered, successive administration 12d.
(3) research to murine melanoma inhibitory action
Drug withdrawal next day execution mice of weighing, dissect and peel off tumor mass, claim tumor weight, by average tumor re-computation tumour inhibiting rate.Press down
Ratio of outflow=[(matched group gross tumor volume-experimental group gross tumor volume)/matched group gross tumor volume] × 100%.
(4) experimental result
Result shows, test the tumour inhibiting rate of 1～9 group be respectively 55.31%, 53.27%, 46.71%, 45.33%,
29.35%, 38.11%, 42.32%, 44.06%, 49.16%.Wherein, the use quality (mg) of PD-1 antibody and bacillus bifidus
Volume (ml) than for 1:1 and 1:2 time, tumor killing effect is optimal.Illustrating, PD-1 monoclonal antibody and bacillus bifidus are used in combination best proportion
For 1:1～2.The ratio of 2:1 is taken second place.Experimental result part table 4.
Each group of inhibition to murine melanoma of table 4
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any is familiar with this
The people of technology, without departing from the spirit and scope of the present invention, can do various change and modification, the therefore protection of the present invention
Scope should be with being as the criterion that claims are defined.
Claims (7)
1. a pharmaceutical composition, it is characterised in that be made up of with bacillus bifidus bacterium solution PD-1 monoclonal antibody.
2. the pharmaceutical composition described in claim 1, it is characterised in that the quality of described PD-1 monoclonal antibody and bacillus bifidus
The volume combination matching of bacterium solution is (1～2) mg:(1～2) ml.
3. the pharmaceutical composition described in claim 2, it is characterised in that described PD-1 monoclonal antibody and the combination of bacillus bifidus
Proportioning is 1mg:1ml.
4. the pharmaceutical composition described in claim 1, it is characterised in that the use content of described PD-1 monoclonal antibody is 2mg/
kg；The viable count of described bacillus bifidus is 1 × 107cfu/mL。
5. claim 1-4 arbitrary described pharmaceutical composition application in preparation treatment cancer drug.
6. apply described in claim 5, it is characterised in that described cancer is pulmonary carcinoma, gastric cancer, hepatocarcinoma, colorectal carcinoma, melanin
Tumor, nephroncus, ovarian cancer, carcinoma of prostate, bladder cancer, breast carcinoma, the esophageal carcinoma, colorectal cancer, nasopharyngeal carcinoma, the cerebral tumor, cervical cancer, blood
Cancer, osteocarcinoma, lymphatic cancer, pancreatic cancer.
7. apply described in claim 6, it is characterised in that described cancer is melanoma.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111096982A (en) * | 2020-02-27 | 2020-05-05 | 上海上药信谊药厂有限公司 | Antitumor composition |
| CN111110852A (en) * | 2020-02-27 | 2020-05-08 | 上海上药信谊药厂有限公司 | Antitumor composition |
| CN111246881A (en) * | 2017-12-14 | 2020-06-05 | 江苏恒瑞医药股份有限公司 | Use of PD-1 antibodies for treating tumors |
| CN111356464A (en) * | 2017-07-05 | 2020-06-30 | 伊夫罗生物科学公司 | Compositions and methods for treating cancer using bifidobacterium animalis subsp |
| CN112641930A (en) * | 2020-12-29 | 2021-04-13 | 江西普瑞森基因科技有限公司 | Combined pharmaceutical composition for resisting colorectal cancer and application thereof |
| CN112805020A (en) * | 2018-09-25 | 2021-05-14 | 国立研究开发法人国立循环器病研究中心 | Antitumor effect potentiator |
| CN113150155A (en) * | 2021-04-22 | 2021-07-23 | 安徽瀚海博兴生物技术有限公司 | anti-PD-L1 humanized monoclonal antibody and application thereof |
| CN113663081A (en) * | 2021-09-16 | 2021-11-19 | 中国药科大学 | Application of oroxylin and PD-1/PD-L1 inhibitor in preparation of liver cancer treatment drug |
| CN114657086A (en) * | 2021-12-27 | 2022-06-24 | 苏州善佰生物技术有限公司 | Preparation method of bifidobacterium and application of bifidobacterium in tumor treatment |
-
2016
- 2016-07-29 CN CN201610615271.7A patent/CN106110322A/en active Pending
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| AYELET SIVAN等: "Commensal Bifidobacterium promotes antitumor immunity and facilitates anti–PD-L1 efficacy", 《SCIENCE》 * |
| NORIHO IIDA等: "Commensal Bacteria Control Cancer Response to Therapy by Modulating the Tumor Microenvironment", 《SCIENCE》 * |
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111356464A (en) * | 2017-07-05 | 2020-06-30 | 伊夫罗生物科学公司 | Compositions and methods for treating cancer using bifidobacterium animalis subsp |
| CN111246881B (en) * | 2017-12-14 | 2023-03-10 | 江苏恒瑞医药股份有限公司 | Use of PD-1 antibodies for treating tumors |
| CN111246881A (en) * | 2017-12-14 | 2020-06-05 | 江苏恒瑞医药股份有限公司 | Use of PD-1 antibodies for treating tumors |
| CN112805020A (en) * | 2018-09-25 | 2021-05-14 | 国立研究开发法人国立循环器病研究中心 | Antitumor effect potentiator |
| CN111110852A (en) * | 2020-02-27 | 2020-05-08 | 上海上药信谊药厂有限公司 | Antitumor composition |
| CN111096982A (en) * | 2020-02-27 | 2020-05-05 | 上海上药信谊药厂有限公司 | Antitumor composition |
| WO2021170138A1 (en) * | 2020-02-27 | 2021-09-02 | 上海上药信谊药厂有限公司 | Anti-tumor composition |
| EP4112065A4 (en) * | 2020-02-27 | 2024-04-17 | Sph Sine Pharmaceutical Laboratories Co Ltd | Anti-tumor composition |
| CN111096982B (en) * | 2020-02-27 | 2023-09-12 | 上海上药信谊药厂有限公司 | Antitumor composition |
| CN112641930A (en) * | 2020-12-29 | 2021-04-13 | 江西普瑞森基因科技有限公司 | Combined pharmaceutical composition for resisting colorectal cancer and application thereof |
| CN113150155A (en) * | 2021-04-22 | 2021-07-23 | 安徽瀚海博兴生物技术有限公司 | anti-PD-L1 humanized monoclonal antibody and application thereof |
| CN113150155B (en) * | 2021-04-22 | 2022-04-08 | 安徽瀚海博兴生物技术有限公司 | anti-PD-L1 humanized monoclonal antibody and application thereof |
| CN113663081A (en) * | 2021-09-16 | 2021-11-19 | 中国药科大学 | Application of oroxylin and PD-1/PD-L1 inhibitor in preparation of liver cancer treatment drug |
| CN114657086A (en) * | 2021-12-27 | 2022-06-24 | 苏州善佰生物技术有限公司 | Preparation method of bifidobacterium and application of bifidobacterium in tumor treatment |
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