CN107029233A - Purposes of the interleukins-8 antibody in endometriosis medicine composition is prepared - Google Patents
Purposes of the interleukins-8 antibody in endometriosis medicine composition is prepared Download PDFInfo
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- CN107029233A CN107029233A CN201610080660.4A CN201610080660A CN107029233A CN 107029233 A CN107029233 A CN 107029233A CN 201610080660 A CN201610080660 A CN 201610080660A CN 107029233 A CN107029233 A CN 107029233A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
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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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
- A61K2039/507—Comprising a combination of two or more separate antibodies
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Abstract
The invention belongs to pharmaceutical technology field, it is related to the new pharmaceutical usage of interleukins-8 antibody, and in particular to interleukins is that IL-10 and IL-17 antibody is used to prepare the purposes in treatment endometriosis (gynecopathy) pharmaceutical composition.Shown through experiment in vitro and internal animal experiment, recombinant il-10 and IL-17A albumen are obviously promoted Endometrial stromal cells (ESC) growth and attacked；IL-10 and IL-17A neutrality antibody drug regimens significantly inhibit the growth of dystopy stove.It is further useful for preparing the medicine for the treatment of endometriosis.
Description
Technical field
The invention belongs to pharmaceutical technology field, it is related to the new pharmaceutical usage of interleukins-8 antibody, and in particular to leucocyte is situated between
Element is that IL-10 and IL-17 antibody is used to prepare the use in treatment endometriosis (gynecopathy) pharmaceutical composition
On the way.Shown through experiment in vitro and internal animal experiment, recombinant il-10 and IL-17A albumen are obviously promoted between endometrium
Cell plastid (ESC) grows and attacked；IL-10 and IL-17A neutrality antibody drug regimens significantly inhibit dystopy stove
Growth.It is further useful for preparing the medicine for the treatment of gynecopathy.
Background technology：
It is a kind of normal prior art discloses endometriosis (abbreviation gynecopathy, Endometriosis, EMS)
The gynecological disease seen, shows as endometrium and appears in other positions (such as ovary, peritonaeum) beyond uterine cavity.Institute
Internal film tissue's generating period bleeding under the influence of the change of ovarian hormone of dystopy is stated, with surrounding annulus hyperblastosis
And Adhesion formation, and then form the ectopic focus (abbreviation dystopy stove) of endometriosis.The whole world is counted according to WHO
EMS number of the infecteds are more than 1.76 hundred million people, and the incidence of disease in Women of childbearing age is about 10%~15%.Studies have shown that EMS
Though being benign lesion, there are the malignant activities such as hyperplasia, plantation infiltration, transfer and recurrence, make obstinate disease.
Clinical practice is shown, pelvic cavity fibrosis, adhesion, the pain and infertile (about 30~50% of gradual exacerbation are presented patient more
EMS patient's secondary infertility), so that have a strong impact on the physical and mental health and quality of life of numerous women, so whole family
With the harmony of society.Although after the research of nearly a century and a half, the pathogenesis for EMS still has too many at present
Unsolved mystery.Medicine and operative treatment are still remedy measures main at present, but recurrence is still that an order is puzzled in the industry
The problem of.Therefore, deeply the pathogenesis of parsing gynecopathy will be to be better understood from this disease in the industry and find more to have
The remedy measures of effect lay the foundation, and have to the health harmony for improving WomanHealth quality and whole family and society important
Strategic importance.
The menses counter-current theory of endometrium is the theory the most recognized by people, however, menses adverse current occurs most
In the menstrual cycle of number women, but only 10%~15% women suffers from EMS；There is research to show endometrial stroma
Cell (ESC) is inherent abnormal related to EMS, including gene unconventionality expression, reaction of the inner membrance to hormone, increases
Nerve density and oxidative stress；Under normal conditions, endometrium countercurrently to pelvic cavity is by the immune system of body
" foreign matter " is identified as to be removed, and in the abdominopelvic cavity of EMS patient, local immune microenvironment is likely occurred
Change, immune cell function not only can not effectively remove the endometrial cell of dystopy extremely, or even help it
In dystopy plantation, growth；Therefore, increasing evidence shows EMS patient's abdominopelvic cavity local immunity micro-loop in recent years
Border plays various immunocytes in indispensable effect, especially dystopy stove in EMS occurrence and development extremely
And the cell factor produced.
The evidence of research accumulation shows that Th17 cells are in various autoimmune diseases and allergic inflammatory diseases
Play key effect；Recently there is research research prompting IL-10+Th17 cells have regulatory function.
Present situation based on prior art, present inventor intends assessing antagonism IL-10 by experimental study+Th17 cells
There is provided interleukins IL-10 for potential values of two key function molecule IL-10 and IL-17 in treatment EMS
It is used to prepare the purposes in treatment endometriosis (gynecopathy) pharmaceutical composition with IL-17 antibody, is EMS
Clinical treatment new method and new approaches are provided.
The content of the invention
It is an object of the invention to provide the new pharmaceutical usage of interleukins-8 antibody, and in particular to interleukins is IL-10
It is used to prepare the purposes in treatment endometriosis (gynecopathy) pharmaceutical composition with IL-17 antibody.
Tested in vitro and resisting in EMS animal models in the present invention by assessing IL-10 and IL-17 neutrality antibodies
Activity in EMS inner membrances ESC growths and invasion and attack；It is IL-10 and IL-17 antibody there is provided a kind of interleukins
The purposes in pharmaceutical composition for preparing treatment EMS.
Research based on early stage of the present invention finds that EMS abdominal cavity of patients liquid has high level Th17 cells and IL-17A,
III-IV phase patients have higher level IL-10 and its IL-10+Th17 cells, are pointed out with progression of disease, in proinflammatory and resistance to
By and deposit and be inclined to tolerance process；The present invention has carried out experiment in vitro and internal animal experiment, as a result shows, restructuring
IL-10 and IL-17A albumen is obviously promoted Endometrial stromal cells (ESC) growth and attacked；IL-10 and IL-17A
Neutrality antibody drug regimen significantly inhibits the growth of dystopy stove；The drug regimen, which is further useful for preparing, treats interior different
The medicine of disease.
Specifically, a kind of biology by combining antagonism cell factor IL-10 and IL-17A of disclosure of the invention is made
With, and then anti-EMS inner membrances ESC growths and invasiveness are played, available for the medicine for preparing treatment EMS.
The purpose of the present invention is achieved through the following technical solutions：
Control and EMS abdominal cavity of patients's liquid are collected, as a result shows that EMS abdominal cavity of patients liquid has high level Th17 cells
And IL-17A, III-IV phase patient have higher level IL-10 and its IL-10+Th17 cells (as shown in Figure 1)；Pass through
In vitro test shows that recombined human IL-17A albumen (10ng/ml, R＆D company) is obviously promoted ESC vigor, suppression
Make its apoptosis, and the Ectopic endometrium ESC for promoting it to breed (as shown in Figure 2), especially EMS patient；Enter one
Step is researched and analysed, and is as a result shown, Recombinant Human IL-10 albumen (10ng/ml, R＆D company) can amplify IL-17A pairs
The facilitation (as shown in Figure 3) of ESC growths；The anti-human IL-10 of use in conjunction (5ug/ml, R＆D company) and IL-17
Neutrality antibody (5ug/ml, R＆D company) substantially suppress Th17 cells to the ESC cell growths of co-cultivation and
The promotion effect (as shown in Figure 4) of invasion and attack；Further, the present invention builds EMS mouse models (as shown in Figure 5),
Anti- mouse IL-10 neutrality antibodies (5ug/ml, R＆D company) and/or rear anti-mouse IL-17A neutrality antibodies is injected intraperitoneally
(5ug/ml, R＆D company), as a result shows, relative to control group, independent IL-10 antibody injection group and independent IL-17A
Antibody injection group mouse dystopy stove product is obviously reduced (as shown in Figure 5)；The anti-mouse IL-10 and IL-17A of use in conjunction
Antibody injection group mouse dystopy stove volume-diminished is significantly (as shown in Figure 5).
It is used for the purposes for treating EMS medicines the invention provides IL-10 and IL-17 antibody；Especially through experiment in vitro
With zoopery confirm, use in conjunction IL-10 and IL-17 neutrality antibody significantly inhibit ESC growth and invasion and attack and
Its dystopy stove size, further, described interleukins are that IL-10 and IL-17 antibody can be used for preparing treatment
Endometriosis (gynecopathy) pharmaceutical composition.
Brief description of the drawings
Fig. 1 is IL-10, IL-17 and IL10+Expression of the Th17 cells in EMS abdominal cavity of patients's liquid,
Wherein, Ctrl:Control group peritoneal fluid,
S(I-II)：I-II phase gynecopathy abdominal cavity of patients's liquid,
S(III-IV)：I-II phase gynecopathy abdominal cavity of patients's liquid,
MFI:Mean fluorescent intensity,
*P<0.05,**P<0.01and***P<0.001；NS:Without significant difference.
Fig. 2 is growth in vitro and the invasion and attack that IL-17A albumen promotes ESC,
Wherein, rhIL-17A：Recombined human IL-17A albumen,
Normal ESCs：Normal endometrium ESC,
Eutopic ESCs:EMS patient Normal endometrium ESC,
Ectopic ESCs:EMS ectopic endometrium stove inner membrance ESC,
Wherein, Cell invasion index of ESCs：ESC invasivenesses index (average cell number under 5 random fields
Amount), using control group as 100, the average ESC of the average ESC quantity/control group of each group invasiveness index=100* experimental groups
Quantity；*P<0.05and**P<0.01；NS:Without significant difference；Original magnification:×200..
Fig. 3 is the facilitation that IL-10 albumen amplification IL-17A grows to ESC,
Wherein, IL-10：Recombinant Human IL-10 albumen treatment group,
IL-17A：Recombined human IL-17A albumen treatment groups,
IL-10+IL-17A：Use in conjunction Recombinant Human IL-10 and IL-17A albumen treatment groups,
*P<0.05,**P<0.01and***P<0.001；NS:Without significant difference, Original magnification:
× 100 or × 400..
Fig. 4 is that IL-10 and IL-17 antibody significantly inhibits Th17 cells to ESC cell growths and the facilitation of invasion and attack,
Wherein, Ctrl:Independent ESC groups,
Th17:The Th17 cells of Differentiation Induction in vitro and ESC co-cultivation groups,
α-IL-10：Anti-human IL-10 neutrality antibodies,
α-IL-17：Anti-human IL-17 neutrality antibodies,
Th17+α-IL-10：Th17 cells and ESC co-cultivations+anti-human IL-10 neutrality antibodies group,
Th17+α-IL-17：Th17 cells and ESC co-cultivations+anti-human IL-17 neutrality antibodies group,
Th17+α-IL-10+α-IL-17：Th17 cells and ESC co-culture+and anti-human IL-10 neutrality antibodies+
Anti-human IL-17 neutrality antibodies group, * P<0.05,**P<0.01and***P<0.001.
Fig. 5 is that use in conjunction IL-10 and IL-17A antibody significantly inhibits the growth of EMS model mice dystopys stove,
Wherein, Ctrl：Control group EMS model mices,
α-IL-10：Anti- mouse IL-10 neutrality antibody treatment groups,
α-IL-17A：Anti- mouse IL-17A neutrality antibody treatment groups,
α-IL-10+α-IL-17：Anti- mouse IL-10 neutrality antibodies+anti-mouse IL-17A neutrality antibody treatment groups.
Embodiment
IL-10, IL-17 and IL10 in the analysis EMS abdominal cavity of patients's liquid of embodiment 1+Th17 cellular levels
Detected, as a result shown using CBA (BD companies) method first, relative to control group, in EMS (I-II phases),
That is disease initial stage, pro-inflammatory cytokine IL-17A is significantly raised；But in EMS (III-IV phases), i.e., after disease
Phase anti-inflammatory cytokines IL-10 significantly raised (as shown in Figure 1A)；Then with flow cytometry, (streaming antibody is purchased
From in Biolegend companies) detection, as a result show, I-II phase EMS abdominal cavity of patients's liquid IL-17A levels are significantly raised,
But with progression of disease, without further elevated trend, but III-IV phase patients have higher level IL-10+Th17 is thin
Born of the same parents (such as Figure 1B, shown in 1C).
The experiment in vitro of embodiment 2 confirms that blocking IL-10 and IL-17 substantially to suppress Th17 cells breeds and attack to ESC
Facilitation
1) IL-17A albumen promotes ESC growth in vitro and invasion and attack
Control internal film tissue, EMS patient's Normal endometrium and Ectopic endometrium tissue are collected, passes through IV collagens
Enzyme (Sigma) digest and it is primary be separately cultured ESC, after then being stimulated 48 hours with recombined human IL-17A albumen, point
Yong not CCK8 (Dong Ren chemical companies) method, double dye (Invitrogen companies) methods of Annexin V-FITC/PI and Matrigel
(BD companies) invasion and attack testing inspection ESC cell viability (as shown in figure Fig. 2A), apoptosis (as shown in Figure 2 B)
With invasiveness (such as Fig. 2 C, shown in 2D), as a result show, recombined human IL-17A is obviously promoted ESC vigor, suppresses it
Apoptosis simultaneously promotes it to attack；
2) the facilitation experiment that IL-10 albumen amplification IL-17A grows to ESC
With Recombinant Human IL-10, IL-17A or use in conjunction IL-10 and IL-17 albumen handle ESC48 hours, together
((as shown in Fig. 3 A, 3B, 3C) is as a result shown independent upper method detection ESC cell viability, apoptosis and invasiveness
IL-10 albumen, which has, to be promoted ESC vigor, suppresses its apoptosis, and promotes it to attack；In addition, Recombinant Human IL-10 egg
White greatly enlarged IL-17A is to the adjustment effect of ESC cell viabilities and apoptosis, but to the regulations of ESC invasivenesses without entering
One step enlarge-effect；
3) IL-10 and IL-17 antibody significantly inhibits Th17 cells and ESC cell growths and the facilitation of invasion and attack is tested
Normal reproduction phase women's human peripheral is collected, peripheral blood is obtained with separation of lymphocytes (it is company that Shenzhen, which reaches section)
Single karyolymph cell (PBMC), then obtains people by magnetic bead (magnetic bead antibody is purchased from Mei Tian girls company) sortingT cell, is then inoculated in that to be coated with anti-CD 3 antibodies (5ug/ml) and anti-CD28 antibody (1ug/ml) (anti-
Body is purchased from eBioscience companies) Tissue Culture Plate in, then with recombined human IL-6 (50ng/ml) and TGF-β
After (5ng/ml) albumen (being purchased from R＆D companies) is stimulated 5 days, the Th17 cells of induction differentiation are collected, by it
Co-cultured with ESC, and it is anti-to add or be added without anti-human IL-10 neutrality antibodies and/or anti-human IL-17 neutralities
Body, and co-culture 48 hours；Then, ibid method, ESC cell viability, apoptosis and invasiveness (such as Fig. 4 A,
Shown in 4B, 4C), as a result show, it is thin to ESC that use in conjunction IL-10 and IL-17 antibody significantly inhibits Th17 cells
Intracellular growth and invasion and attack have facilitation.
Use in conjunction IL-10 and the IL-17 neutrality antibody of embodiment 3 significantly inhibits EMS mouse dystopys stove growth animal
Experiment
(1) EMS mouse models are built：Healthy 7 week old female C57B/L6 mouse (Shanghai Si Laike experimental animals
Co., Ltd), art the last week is induced 1 time with estrogen intramuscular injection, and opening abdomen under then anaesthetizing is derived from body bilateral uterine,
4 parts are divided into, itself four quadrant of peritonaeum, postoperative 14 days conventional treatment mouse, EMS mouse models are sewn on respectively
It is successfully established (as shown in Figure 5A)；
(2) 4 days after EMS mouse modelings, experimental group gives in anti-mouse mouse IL-10 neutrality antibodies, anti-mouse IL-17A
With two kinds of antibody of property antibody or use in conjunction (antibody equal 5ug/ml, cumulative volume 15ul/g), and in strengthening one after 4 days
It is secondary, 2 times altogether；Control group gives solvent (PBS) processing；14 days conventional treatment mouse after same modeling, take different
Position stove, as a result shows, anti-mouse IL-10 antibody or the contracting of anti-mouse IL-17 antibodyome dystopys stove is used alone relative to control group
It is small；Use in conjunction IL-10 and IL-17 neutrality antibody significantly inhibits the growth of EMS mouse dystopys stove, with significantly strong
Anti- EMS effects.
The present invention is confirmed through in vitro and in vivo result of the test, can substantially be pressed down using the combination of IL-10 and IL-17 neutrality antibodies
ESC growths processed, invasion and attack and the growth of dystopy stove.
Claims (7)
1. interleukins IL-10 and IL-17 antibody are for preparing the use in treatment endometriosis medicine composition
On the way.
2. the purposes as described in claim 1, it is characterised in that interleukins IL-10 and IL-17 antibody promote uterus
Inner membrance interstitial cell growth and invasion and attack.
3. the purposes as described in claim 1, it is characterised in that interleukins IL-10 and IL-17 antibody suppress dystopy
The growth of stove.
4. interleukins IL-10 antibody is for preparing the purposes in treatment endometriosis medicine.
5. interleukins IL-17 antibody is for preparing the purposes in treatment endometriosis medicine.
6. interleukins IL-10 and IL-17 antibody are for preparing the purposes in clinical assessment EMS progress methods.
7. the purposes as described in claim 6, it is characterised in that by detecting IL-10 in peritoneal fluid+Th17 cellular levels
Progress extent for assessing EMS diseases.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610080660.4A CN107029233A (en) | 2016-02-04 | 2016-02-04 | Purposes of the interleukins-8 antibody in endometriosis medicine composition is prepared |
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|---|---|---|---|
| CN201610080660.4A CN107029233A (en) | 2016-02-04 | 2016-02-04 | Purposes of the interleukins-8 antibody in endometriosis medicine composition is prepared |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110862963A (en) * | 2019-11-27 | 2020-03-06 | 沣潮医药科技（上海）有限公司 | Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility-related diseases |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013045404A2 (en) * | 2011-09-28 | 2013-04-04 | Bayer Intellectual Property Gmbh | Inhibition of the effect of interleukin 1 beta in order to treat endometriosis |
-
2016
- 2016-02-04 CN CN201610080660.4A patent/CN107029233A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013045404A2 (en) * | 2011-09-28 | 2013-04-04 | Bayer Intellectual Property Gmbh | Inhibition of the effect of interleukin 1 beta in order to treat endometriosis |
Non-Patent Citations (4)
| Title |
|---|
| SCHWAGER 等: "The antibody-mediated targeted delivery of interleukin-10 inhibits endometriosis in a syngeneic mouse model", 《HUMAN REPRODUCTION》 * |
| SUEN 等: "Serum Level of IL-10 Is Increased in Patients with Endometriosis, and IL-10 Promotes the Growth of Lesions in a Murine Model", 《THE AMERICAN JOURNAL OF PATHOLOGY》 * |
| 常凯凯: "子宫内膜异位灶微环境IL-10+Th17细胞的形成及作用机制", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
| 苏春兰: "桂枝茯苓丸对子宫内膜异位症患者血清中IL-10和IL-17的影响", 《北方医学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110862963A (en) * | 2019-11-27 | 2020-03-06 | 沣潮医药科技（上海）有限公司 | Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility-related diseases |
| CN110862963B (en) * | 2019-11-27 | 2021-08-27 | 沣潮医药科技（上海）有限公司 | Application of decidua NK cells and cell subsets thereof in preparation of medicines for treating infertility-related diseases |
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Application publication date: 20170811 |