CN102058575B - Application of serine protease inhibitor to anti-embryo implantation and anti-tumor aspects - Google Patents
Application of serine protease inhibitor to anti-embryo implantation and anti-tumor aspects Download PDFInfo
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Abstract
The invention provides application of a serine protease inhibitor to anti-embryo implantation and anti-tumor aspects, and in particular discloses application of the serine protease inhibitor (an active derivative or pharmaceutically acceptable salt thereof) such as 4-(2-aminoethyl) phenylsulfonyl fluoride, gabexate mesilate and the like to the anti-embryo implantation and anti-tumor aspects. The invention also provides the application of the serine protease inhibitor to preparation of a composition or an appliance for suppressing embryo implantation or tumor cell growth.
Description
Technical field
The invention belongs to biotechnology and fertility adjusting field, specifically, the present invention relates to serpin and the application at anti-embryo nidation and anti-tumor aspect thereof.
Background technology
To be that mammal and the mankind are distinctive a kind ofly can determine Pregnancy Success whether reproductive physiology step to embryo nidation (implantation), is also the desirable link of carrying out genital regulating.Implantation process generally comprises embryo's stage such as location, adhesion and intrusion of film in uterus, relates to growth, differentiation and the migration of the cell at mother-tire interface, and the cell physiological mechanism of the tool general character such as intercellular mutual identification, adhesion, signal transduction.Simultaneously, Invasion and Metastasis is also the biological behaviour of the tool feature of malignant cell, and this behavior is very similar to the physiological process of embryo nidation, all show as the fast breeding of cell and differentiation, lasting angiogenesis, formation, the intrusion to surrounding tissue etc. of local immunity tolerance status.
Embryo is with respect to parent, and Shi Yigeban allosome tissue, is subject to parent and repels, and can only within a specific time period, (i.e. " implantation window ") could be received by parent endometrial tissue.What the completing of embryo nidation process depended on parent uterus and embryo synchronizes the formation of growth, parent endometrium receptivity and " implantation window " and the formation of parent uterine cancer cell local immunity toleration, relates to the adjusting of the propagation of cell and differentiation, Effects on local immunological functions.Female, the effect of progestogen in implantation is very clear and definite, based on them, the effect in reproductive process grows up existing steroid contraceptive.But because female, progestogen are endocrine hormones, its effect is general, it not also therefore optimal fertility regulatory factor.
In the process of embryo nidation, embryo's syneytiotrophoblast is secreted a series of protease, by the endometrial extracellular matrix of degrading (extracellular matrix, ECM), promotes endometrial reconstruct.Extracellular matrix is some macromolecular substances that cell produces, and comprises collagen protein, non-collagen sugar albumen and proteoglycan three major types.Most of compositions of people's endometrium ECM change along with the variation of the different phase of menstrual cycle, and relevant to implantation is secretion mid-term and decidua.Trimester blastocyst sticks to the endometrium of decidua.When implantation, zona pellucida dissolves disappearance completely, and first the trophoderm of inner cell mass side contacts with endometrium, extracellular proteinase, and at breach of inner membrance stripping, then blastocyst is absorbed in breach, gradually by embedding wherein.For the embryo after implantation can be survived, syneytiotrophoblast will destroy blood capillary and the vein blood vessel of decidua and penetrate spiral artery, and induction spiral artery becomes the blood vessel of the high blood flow of lower resistance, to meet the nutritional need of body early embryo.
For various reasons, people's pair factor relevant with embryo nidation also known little about it, and at present known inhibition embryo nidation is also little, and therefore this area suppresses the material of embryo nidation in the urgent need to developing new can be used for.
Summary of the invention
The object of this invention is to provide a kind of new can be used for and suppress the inhibitor of embryo nidation and the purposes in fertility regulates thereof.
In a first aspect of the present invention, a kind of purposes of serpin is provided, described serpin is selected from lower group: 4-(2-aminoethyl) benzene sulfonyl fluorine or its reactive derivative or its pharmaceutically acceptable salt; Gabexate or its reactive derivative or its pharmaceutically acceptable salt, wherein, described inhibitor is used to compositions or the utensil that preparation suppresses embryo nidation or inhibition tumor cell growth.
In another preference, described compositions or utensil are by the compositions of vagina administration or utensil.
In another preference, described inhibitor is the 4-shown in following formula (2-aminoethyl) benzene sulfonyl fluorine hydrochlorate
Or described reactive derivative is to have the derivant that serine protease suppresses active 4-(2-aminoethyl) benzene sulfonyl fluorine.
In another preference, described inhibitor is the gabexate mesilate shown in following formula
Or described reactive derivative is to have the derivant that serine protease suppresses active gabexate.
In another preference, described compositions is pharmaceutical composition.
In another preference, described pharmaceutical composition is the compositions that suppresses embryo nidation.
In another preference, described pharmaceutical composition is contraceptive; Or described utensil is contraceptive device.
In another preference, described contraceptive device comprises (but being not limited to): pessary or intrauterine device etc.
In another preference, described contraceptive comprises vagina gel.
In another preference, described inhibitor is used to compositions or the utensil that preparation (simultaneously) suppresses embryo nidation and inhibition tumor cell growth.
In a second aspect of the present invention, a kind of utensil that suppresses embryo nidation is provided, described utensil is the contraceptive device for women, it is characterized in that, described contraceptive device also comprises that wherein said serpin is selected from lower group: 4-(2-aminoethyl) benzene sulfonyl fluorine or its reactive derivative or its pharmaceutically acceptable salt for the releasing parts at vagina or intrauterine release serpin; Gabexate or its reactive derivative or its pharmaceutically acceptable salt.
In another preference, described releasing parts is arranged at or is fixed on the body of contraceptive device.
In another preference, described releasing parts is slow release layer.
In another preference, described utensil is that contraceptive device comprises (but being not limited to): pessary or intrauterine device etc.
In a third aspect of the present invention, a kind of contraceptive device of non-therapeutic purposes is provided, the method comprises: use serpin to the female mammal of needs contraception, wherein said serpin is selected from lower group: 4-(2-aminoethyl) benzene sulfonyl fluorine or its reactive derivative or its pharmaceutically acceptable salt; Gabexate or its reactive derivative or its pharmaceutically acceptable salt.
In another preference, described using is local application, is more preferably intravaginal or intrauterine local application.
In a fourth aspect of the present invention, a kind of purposes of serpin is provided, wherein said serpin is selected from lower group: 4-(2-aminoethyl) benzene sulfonyl fluorine or its reactive derivative or its pharmaceutically acceptable salt; Gabexate or its reactive derivative or its pharmaceutically acceptable salt, described inhibitor is used as suppressing the inhibitor of embryo nidation.
Other side of the present invention, because technology is herein open, is apparent to those skilled in the art.
Brief description of the drawings
Fig. 1 has shown Chinese People's Anti-Japanese Military and Political College's Mus implantation effect of 2 kinds of serpins (AEBSF and GM) vagina administrations.
Fig. 2 has shown the anti-mice embryonic implantation effect of AEBSF cornua uteri administration.AEBSF group (the one-sided cornua uteri of medication 30-3000 μ g/) and the result of normal saline (PS) matched group relatively show, AEBSF dose dependent ground inhibition mice embryonic implantation.
Fig. 3 has shown the impact of AEBSF on growth of tumour cell.In figure, show, the cell survival rate of A549 lung cancer cell line and the squamous cell carcinoma strain of HCC-94 uterus reduces gradually along with the rising of AEBSF Drug level.
Fig. 4 has shown the impact of gabexate mesilate on growth of tumour cell.In figure, show, in high dose situation, gabexate mesilate suppresses the growth of A549 cell and HCC-94.
Fig. 5 has shown the inhibition test result of contrast inhibitor Antipain cornua uteri administration to mice implantation.In figure, show, the result of Antipain processed group (the one-sided cornua uteri of medication 0,300 or 1000 μ g/) relatively shows, Antipain processes mice embryonic implantation without significant impact.
Detailed description of the invention
The inventor finds by further investigation, be surprised to find that first some serpin or its reactive derivative (comprising pharmaceutically acceptable salt) can suppress embryo nidation, and this inhibitory action is reversible, be therefore particularly suitable for this compounds for suppressing embryo nidation.In addition, these serpins also have inhibition tumor cell growth.
Particularly, the inventor's research shows, 4-(2-aminoethyl) benzene sulfonyl fluorine and gabexate can very effectively and safely suppress embryo's implantation.
The protease working in embryo nidation, can be divided into three major types according to their catalytic activity: matrix metalloproteinase, cysteine proteinase and serine protease.Wherein, the main biological function of MMP is degraded ECM and modifies some important bioactive molecules, in cell migration, invasion and attack, proliferation and apoptosis process, plays a significant role.In the known MMP molecule relevant to implantation, the effect substrate of MMP-1 (collagenase) is multiple collagen-type; The substrate of MMP-3 (substrate hydrolytic enzyme) is proteoglycan, fibronectin, laminin，LN, IV Collagen Type VI etc.; The substrate of MMP-2 (gelatin enzyme A) and MMP-9 (Gelatinase B) comprises multiple collagen, gelatin, fibronectin, laminin，LN etc.
In biology, to serine protease most study.Their common feature is to have serine residue in its active center, the strongest at neutral pH environment activity.The distribution of serine protease is very wide, kind is also maximum, has 20 Duo Ge families, in the physiology such as the Invasion and Metastasis of immune response, tumor cell, blood coagulation, wound healing and pathological process, plays a significant role.The serine protease relevant to embryo nidation of having found at present has plasma urokinase-type plasminogen activator (uPA), kallidinogenase, trypsinlike enzyme, implantation serine protease-1 (ISP-1) and-2 (ISP-2) etc., all belong to the S1 family in Chymotrypsin, mostly participate in the reconstruct of ECM, subparticipation regulates the activity of MMP.
Murine interleukin secretion serine stretch protein enzyme inhibition factor (secretory leukocyte proteaseinhibitor, SLPI) implantation window phase down-regulated expression in film in uterus, it can be invaded in endometrium process and have an effect embryo by suppressing ISP activity.
The discoveries such as Monzon, the u-PA that people's trophocyte produces has obvious protein dissolution activity in vitro, and the fibronectin splicing variants outside energy degradation of cell plays a significant role in embryo nidation.Recently, two kinds of serine proteases that O ' Sullivan etc. find, ISP1 and ISP2 also may play a significant role in embryo nidation.The inventor finds after deliberation, will resist ISP2 antibody to inject uterine cavity, can suppress embryo nidation, and be dosage effect.4-(2-aminoethyl) benzene sulfonyl fluorine and gabexate (or its reactive derivative or its pharmaceutically acceptable salt) inhibitory action to embryo nidation and the inhibitory action of anti-ISP2 antibody may have similarity.
The inventor's research prompting, the mechanism that 4-(2-aminoethyl) benzene sulfonyl fluorine and gabexate (or its reactive derivative or its pharmaceutically acceptable salt) suppress embryo nidation may be to disturb embryo's implantation by the activity of inhibition serine protease (wherein may comprise tPA, the various serine proteases of the Chymotrypsin S1 families such as uPA, kallidinogenase, ISP1, ISP2).Certainly, should understand protection scope of the present invention and not be subject to the impact of this mechanism.
In addition the also growth of the growth of inhibition tumor cell, especially lung carcinoma cell and uterus carcinoma cell effectively of 4-(2-aminoethyl) benzene sulfonyl fluorine and gabexate (or its reactive derivative or its pharmaceutically acceptable salt).
As used herein, " serpin " or " active component " is used interchangeably, and refers to 4-(2-aminoethyl) benzene sulfonyl fluorine or its reactive derivative or its pharmaceutically acceptable salt; And/or gabexate or its reactive derivative or its pharmaceutically acceptable salt.
As used herein, term " 4-(2-aminoethyl) benzene sulfonyl fluorine " refers to following formula: compound:
The English name of 4-(2-aminoethyl) benzene sulfonyl fluorine: 4-(2-Aminoethyl) benzenesulfonylfluoride (abbreviation: AEBSF); Molecular formula: C
8h
10nSO
2f.AEBSF is a kind of serpin, can suppress trypsin, Chymotrypsin, fibrinolysin, thrombin and kallikrein.
As used herein, term " gabexate " refers to following formula: compound:
Gabexate (Gabexate mesilate, referred to as GM) is a kind of serpin of synthetic.Up to now, the unexposed inhibitory action of gabexate to pulmonary carcinoma and uterus carcinoma of crossing.
As used herein, term " the compounds of this invention " and " inhibitor of the present invention " are used interchangeably, and refer to 1. AEBSF and reactive derivative thereof and/or 2. GM and reactive derivative thereof, and this term also comprises the pharmaceutically acceptable salt of AEBSF and GM.
The present invention also comprises the reactive derivative of AEBSF and GM.As used herein, term " reactive derivative " refers to and has substantially kept the serine protease of AEBSF and GM to suppress active derivant.
As used herein, term " pharmaceutically acceptable salt " refers to inorganic acid addition salt or the organic acid addition salt of relatively nontoxic the compounds of this invention.These salt can be in the last separation of compound and purification process in situ preparation, or the compound of purification is reacted with its free alkali form with suitable organic or inorganic acid, then the salt of formation is separated and made.Exemplary salt comprises hydrobromate, hydrochlorate, sulfate, sulphite, acetate, oxalates, valerate, oleate, palmitate, stearate, laruate, borate, benzoate, lactate, phosphate, toluate, citrate, maleate, fumarate, succinate, tartrate, benzoate, mesylate, gluconate, Lactobionate and lauryl sulfonate etc.They can comprise the cation based on alkali and alkaline earth metal ions, as sodium, lithium, potassium, calcium, magnesium etc., and without toxic amine, quaternary amine and amine cation, include but not limited to: amine, tetramethyl amine, tetraethyl amine, methylamine, dimethylamine, trimethylamine, triethylamine, ethamine etc.
For AEBSF, a kind of preferred salt is inorganic acid addition salt, particularly preferably is the hydrochlorate of AEBSF, and its structural formula is as follows:
The molecular formula of 4-(2-aminoethyl) benzene sulfonyl fluorine hydrochlorate: C
8h
10nSO
2fHCl; Molecular weight is 239.7.This compound water soluble, more stable in the aqueous solution of pH < 7, can store 6 months in 4 DEG C; In the aqueous solution of pH > 7, unstable, should prepare in use.
For GM, a kind of preferred salt is organic acid addition salt, particularly preferably is the mesylate of GM, and its structural formula is as follows:
As used herein, term " mammal " comprises the animals such as (but being not limited to) people, monkey, cattle, sheep, pig, rabbit, Mus, cat, Canis familiaris L..Preferred mammal comprises Primate, rodent, Canis animals and felid and various domestic animal and house pet etc.
Fertility regulates compositions
The present invention also provides a kind of fertility to regulate compositions, the compounds of this invention that it contains safe and effective amount and pharmaceutically acceptable carrier or excipient.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Pharmaceutical preparation should match with administering mode.Pharmaceutical composition of the present invention can be made into injection form, for example, be prepared by conventional method with normal saline or the aqueous solution that contains glucose and other adjuvant.Pharmaceutical composition such as Tablet and Capsula, can be prepared by conventional method.Pharmaceutical composition should be manufactured as injection, solution, Tablet and Capsula under aseptic condition.The dosage of active component be contraception effective dose, for example every day approximately 1 microgram/kg body weight-Yue 10 mg/kg body weight.In addition, compound of the present invention also can use together with other contraceptives.
The compounds of this invention or its pharmaceutically acceptable salt can be oral, parenteral (intravenous, intramuscular or subcutaneous), topical (intravaginal or intrauterine administration, for example, as powder, ointment or drop).A kind of particularly preferred method of application is local application, is more preferably intravaginal or intrauterine local application.
Solid dosage forms for oral administration comprises capsule, tablet, pill, powder and granule.In these solid dosage formss, reactive compound mixes with at least one conventional inert excipient (or carrier), as sodium citrate or dicalcium phosphate, or mix with following compositions: (a) filler or bulking agent, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binding agent, for example, hydroxy methocel, alginate, gelatin, polyvinyl pyrrolidone, sucrose and arabic gum; (c) wetting agent, for example, glycerol; (d) disintegrating agent, for example, agar, calcium carbonate, potato starch or tapioca, alginic acid, some composition silicate and sodium carbonate; (e) retarding solvent, for example paraffin; (f) absorb accelerator, for example, quaternary ammonium compound; (g) wetting agent, for example spermol and glyceryl monostearate; (h) adsorbent, for example, Kaolin; (i) lubricant, for example, Talcum, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulphate, or its mixture.In capsule, tablet and pill, dosage form also can comprise buffer agent.
Solid dosage forms is prepared as tablet, sugar pill, capsule, pill and granule can adopt coating and shell material, as casing and other material well known in the art.They can comprise opacifying agent, and, in the mode that in this compositions, the release of reactive compound or compound can postpone certain part in digestive tract, discharge.The example of adoptable embedding component is polymeric material and Wax.If desired, reactive compound also can with above-mentioned excipient in one or more form microencapsulation form.
Liquid dosage form for oral administration comprises pharmaceutically acceptable emulsion, solution, suspension, syrup or tincture.Except active ingredient beyond the region of objective existence, liquid dosage form can comprise the conventional inert diluent adopting in this area, as water or other solvent, solubilizing agent and emulsifying agent, example is known, the mixture of ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3 butylene glycol, dimethyl formamide and oil, particularly Oleum Gossypii semen, Oleum Arachidis hypogaeae semen, maize embryo oil, olive oil, Oleum Ricini and Oleum sesami or these materials etc.
Except these inert diluents, compositions also can comprise auxiliary agent, as wetting agent, emulsifying agent and suspending agent, sweeting agent, tender taste agent and spice.
Except active ingredient beyond the region of objective existence, suspension can comprise suspending agent, for example, and the mixture of ethoxylation isooctadecane alcohol, polyoxyethylene sorbitol and Isosorbide Dinitrate, microcrystalline Cellulose, aluminium methoxide and agar or these materials etc.
Compositions for parenteral injection can comprise physiologically acceptable aseptic moisture or anhydrous solution, dispersion liquid, suspension or emulsion, and for being again dissolved into aseptic Injectable solution or the sterilized powder of dispersion liquid.Suitable moisture and nonaqueous carrier, diluent, solvent or excipient comprises water, ethanol, polyhydric alcohol and suitable mixture thereof.
The dosage form that is used for the compounds of this invention of topical comprises powder, ointment, powder or propellant.Active component under aseptic condition with physiologically acceptable carrier and any antiseptic, buffer agent, or the propellant that may need is if desired mixed together.
While making pharmaceutical composition, that the compounds of this invention of safe and effective amount is applied to mammal, wherein this safe and effective amount is conventionally at least about 1 microgram/kg body weight, and be in most of the cases no more than approximately 10 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered the factors such as the health status of route of administration, use object, and these are all within skilled practitioners skill.
Contraceptive device
The present invention also provides a kind of utensil that suppresses embryo nidation, the contraceptive device of described utensil for containing AEBSF and/or GM (or its reactive derivative or its pharmaceutically acceptable salt).
In contraceptive device of the present invention, comprise for the releasing parts at vagina or intrauterine release AEBSF and/or GM (or its reactive derivative or its pharmaceutically acceptable salt).In another preference, described contraceptive device is pessary or intrauterine device.
Should be understood that described releasing parts can be independent, also can combine.For example, releasing parts can be an independent releasing member or the combination type releasing member combined with miscellaneous part, or is bonded in or is integrated in the releasing layer of miscellaneous part.
A kind of optimal way is on conventional pessary or intrauterine device, to add a releasing layer (for example slow release layer), this releasing layer can discharge in intravaginal or in utero AEBSF and/or GM (or its reactive derivative or its pharmaceutically acceptable salt) within a period of time, thereby suppresses embryo nidation.
Conventionally, the releasing member in a contraceptive device should contain 5-5000 milligram, more preferably 10-2500 milligram, the best AEBSF of 20-1000 milligram and/or GM or its reactive derivative or its pharmaceutically acceptable salt.
Major advantage of the present invention is,
(a) AEBSF and/or GM (or its reactive derivative or its pharmaceutically acceptable salt) have obvious inhibitory action to embryo nidation;
(b) by intravaginal or intrauterine topical AEBSF and/or GM (or its reactive derivative or its pharmaceutically acceptable salt), there is very high safety good.
(c) be reversible to the inhibitory action of embryo nidation.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.
Embodiment 1
The inhibitory action of AEBSF to embryo nidation
(1) raising of laboratory animal and copulation;
The bull ICR mice in 8～10 week age (35～40 grams of body weight) and female ICR mice (25～30 grams of body weight) are mated to raising in 2: 1 ratios, morning next day is by checking that female mouse vagina bolt is to judge whether copulation, to see that bolt same day is as pregnant D1 days, and by with a collection of detect pregnant D1 days mices divide into groups at random.
(2) preparation of injection solution:
The compound using is the hydrochlorate of AEBSF, and its structural formula is as follows:
1, AEBSF high dose group: 200 μ g/ μ l (preparing with normal saline),
2, dosage group in AEBSF: 20 μ g/ μ l (preparing with normal saline),
3, AEBSF low dose group: 2 μ g/ μ l (preparing with normal saline).
(3) inject time and method:
In 8:00～10:00 in the pregnant D3 days mornings, the position from cornua uteri upper end, mice both sides near ovary, with microsyringe, by the injection solution injection cavity of uterus of preparation preheating at 35 DEG C in advance, each cornua uteri injects 15 μ l solution.With injecting identical solution in two cornua uteris of a mice, separate groups of mice is by setting the different injection solution of injection.Put to death mice in pregnant D8 days (the visible embryo of naked eyes) 9:00 in the morning, complete uterine cancer cell and ovary are peeled off, the embryo number to every Side cornua uteri and corpus luteum number (being number of eggs ovulated) count.
(4) result:
Compared with matched group, suppression ratio is 32%, and the suppression ratio of basic, normal, high dosage group is respectively 16%, 53%, 87%; There is obvious dosage effect.
The inhibitory action of table 1AEBSF to embryo nidation
| Group | Injection solution | Cornua uteri number (n) | Corpus luteum number/cornua uteri (X ± SD) | Embryo number/cornua uteri (X ± SD) | Suppression ratio |
| Matched group | Normal saline | 10 | 6.1±1.7 | 5.5±1.6 | |
| Low dose group | 2μg/μl AEBSF | 10 | 5.8±2.3 | 4.4±2.0 | 16％ |
| Middle dosage group | 20μg/μl AEBSF | 12 | 5.7±2.1 | 2.4±2.2 | 53％ |
| High dose group | 200μg/μl AEBSF | 20 | 5.8±2.6 | 0.7±0.9 | 87％ |
Embodiment 2
The reversibility of AEBSF antifertility action
The antifertility action of considering contraceptive must be reversible, and by zoopery, the reversibility of the anti-implantation effect to AEBSF has been carried out observation analysis in the present embodiment.Method is as follows:
Same batch of female adult mice is divided into two groups, and in gravidic pregnant the 3rd day for the first time, the AEBSF by normal saline and 200 μ g/ μ l concentration processed respectively, observed its pregnancy outcome; Place after one month, copulation again, does not give any processing, observes its pregnancy outcome.
Result is as shown in table 2.Result shows, the AEBSF of 200 μ g/ μ l concentration has obvious antifertility action, and this effect reversible (table 2)
The reversibility result of study of table 2.AEBSF antifertility action
Embodiment 3
Containing the pharmaceutical composition of 4-(2-aminoethyl) benzene sulfonyl fluorine hydrochlorate
In the present embodiment, the gel of preparation one vagina administration.Method is as follows:
18 grams of Poloxamer127 (being medical auxiliary materials Technology Co., Ltd. purchased from upper Hydron) and 0.65 gram of F68 (being medical auxiliary materials Technology Co., Ltd. purchased from upper Hydron) are placed in to agitator, add 60 milliliters of 0.4M acetate buffer solutions, stir evenly at 23-25 DEG C, mixing speed 100-300rpm, it is dissolved completely, the about 3-4 hour of process, the benzoic acid (being medical auxiliary materials Technology Co., Ltd. purchased from upper Hydron) of 10 milliliters of glycerol, 0.5 milliliter of polydimethylsiloxane and 0.1 milliliter, stir again 1-2 hour, hold over night, centrifugal 15min, removes bubble.Finally, being made into total amount with 0.4M acetate buffer solution is the gel-type vehicle of 100 milliliters.Measure pH and the viscosity (room temperature 23-25 DEG C) of gel.Result pH is 3.5.With NDJ-1 type rotary viscosimeter (being medical auxiliary materials Technology Co., Ltd. purchased from upper Hydron), mensuration viscosity is 11-15PaS..
In 10ml gel-type vehicle, add 5g serpin, under room temperature, stir and become the gel preparation that final concentration is 0.5g/ml, at 4 DEG C, store for future use.
This gel can be used for vagina administration.
Embodiment 4
Containing the contraceptive device of 4-(2-aminoethyl) benzene sulfonyl fluorine hydrochlorate
Apply gel prepared by one deck embodiment 3 in conventional pessary inner side or outside in pessary, after solidifying, this gel forms a releasing layer that contains 4-(2-aminoethyl) benzene sulfonyl fluorine hydrochlorate, thereby makes the contraceptive device containing 4-(2-aminoethyl) benzene sulfonyl fluorine hydrochlorate.
At embryo nidation in earlier stage, by the uterus such as pessary, intrauterine device local medicine-applying system, discharge the AEBSF of doses in uterus, can suppress embryo's implantation, thereby reach the object avoiding conception and controling birth.
Embodiment 5
Serpin suppresses the effect of rat implantation
5.1 material
4-(2-aminoethyl) benzene sulfonyl fluorine (4-(2-aminoethyl) benzenesulfonyl fluoridehydrochloride, AEBSF), purchased from Amresco company.Gabexate mesilate (Gabexatemesylate, GM), purchased from Jinan Gao De Pharmaceutical Technology Co., Ltd.
SD rat, 4 monthly ages, purchased from Shanghai Si Laike laboratory animal company.
The composition of gel is active component (AEBSF or GM) and substrate, and the main component of its mesostroma is POLOXAMERS.
5.2 experimental technique
Rat male and female mate, female: hero is to mate at 1: 1.Second day female Mus vaginal smear, micro-Microscopic observation finds to be decided to be pregnant the 1st day (pregnant d1) same day of sperm.
Intrauterine Injection group, in pregnant d4 morning, after anesthesia, is injected uterine cavity from cornua uteri position, every Aconitum carmichaeli Debx. palace subscript enters 20mg or 10mg medicine (being dissolved in 50 μ l normal saline), injects respectively bilateral uterus.With solvent, and 50 μ l physiologic saline for substitute AEBSF, same behaviour act as contrast.
Vagina administration group is injected 100mg/ medicine (being dissolved in 0.2ml gel-type vehicle), pregnant d3 and 2 administrations in the d4 morning to intravaginal.With not making blank containing 200 μ l gel-type vehicles of medicine.
The animal of AEBSF and 2 kinds of drug study of gabexate is grouped as follows:
1) normal saline uterine cavity matched group, 3
2) AEBSF Intrauterine Injection group, 3
3) gabexate Intrauterine Injection group, 3
4) gel-type vehicle vagina administration matched group, 7
5) AEBSF vagina administration group, 5
6) gabexate vagina administration group, 3
The rat of each experimental group, in pregnant d10 morning, puts to death rat, takes out uterus and ovary, and live tire number, stillborn fetus number of counting respectively counted the corpus luteum number of ovary simultaneously, and the count results between is more on the same group analyzed the Antiimplantation Action of 2 kinds of medicines.
5.3 interpretation of result
Intrauterine Injection high dose group, i.e. the dosage of the one-sided cornua uteri of 20mg AEBSF/, 3 rats are all dead.
AEBSF group (pregnant d3 and the each 100mg/200 μ of pregnant d4 l vagina administration) and the result of normal saline (PS) matched group relatively show, AEBSF significantly reduces embryo's implantation number (p=0.03).Gabexate processed group (pregnant d3 and the each 100mg/200 μ of pregnant d4 l vagina administration) is not found implantation embryo, compares significant difference (p=0.00) (Fig. 1) with PS group.
These results show, AEBSF and GM all can significantly suppress the embryo nidation of rat.
Embodiment 6
Serpin suppresses the effect of mice implantation
6.1 material
ICR mice, age in 8-10 week, purchased from Shanghai western pul-Bi Kai laboratory animal company limited.
6.1 experimental technique
Mice male and female mate, female: hero is to mate at 2: 1.Within second day, female Mus vagina is found cloudy bolt, is decided to be pregnant the 1st day (pregnant d1) same day.
Intrauterine Injection group, in pregnant d4 morning, after anesthesia, is injected uterine cavity from cornua uteri position, every Aconitum carmichaeli Debx. palace subscript enters 30 μ g, 300 μ g, 1000 μ g or 3000 μ g medicines (being dissolved in 15 μ l normal saline), injects respectively bilateral uterus.With solvent, i.e. 15 μ l physiologic saline for substitute AEBSF, same behaviour act as contrast.
Vagina administration group is injected 10mg/ time to intravaginal, medicine (being dissolved in 0.2ml gel-type vehicle), pregnant d3 and 2 administrations in the d4 morning.With not making blank containing 200 μ l gel-type vehicles of medicine.
The animal of AEBSF drug study is grouped as follows:
(1) normal saline uterine cavity matched group, 6
(2) AEBSF Intrauterine Injection group (30 μ g/ cornua uteri), 6
(3) AEBSF Intrauterine Injection group (300 μ g/ cornua uteri), 5
(4) AEBSF Intrauterine Injection group (1000 μ g/ cornua uteri), 7
(5) AEBSF Intrauterine Injection group (3000 μ g/ cornua uteri), 5
The mice of each experimental group, in pregnant d8 morning, puts to death mice, takes out uterus and ovary, and live tire number, stillborn fetus number of counting respectively counted the corpus luteum number of ovary simultaneously, and the count results between is more on the same group analyzed the Antiimplantation Action of 2 kinds of medicines.
6..3 interpretation of result
Intrauterine injection administration AEBSF, dosage is the one-sided cornua uteri of 30-3000 μ g/, presentation of results AEBSF can suppress mice embryonic implantation in significance ground, and has significant dose dependent.And corpus luteum is influenced not remarkable.Result is as shown in Fig. 2 and Biao 3-4.
Table 3 Mouse Uterus medication result one factor analysis of variance
| Quadratic sum (sum of square) | Df | Mean square | F | Significance (Sig.) | |
| Between group | 426.982 | 4 | 106.745 | 44.026 | 0.000 |
| In group | 58.190 | 24 | 2.425 | ||
| Add up to | 485.172 | 28 |
Comparison between each group of table 4 Mouse Uterus medication result
* in 0.05 level, there is significant difference
* Vehicle represents the blank group of 15 μ l normal saline.
Embodiment 7
The growth of serine protease inhibition tumor cell
7.1 material
A549 lung cancer cell line and the squamous cell carcinoma strain of HCC-94 uterus.
7.2 assay method
(1) be inoculated in 96 well culture plates with the density of 5000 cells in every hole, 37 DEG C, 5%CO
2in incubator, be cultured to cell attachment, add serpin, every kind of inhibitor is established 5 concentration, and each concentration repeats 3 holes.
(2) 37 DEG C, 5%CO
2in incubator, cultivate 48h, every hole adds MTT (5mg/ml) 20 μ l, continues to cultivate 4h.
(3) inhale and abandon culture medium, every hole adds 150 μ L DMSO, vibrates to crystallization and fully dissolves.
(4) detect the A570nm light absorption value of each group of cell by microplate reader, with the cell hole of without inhibitor as a control group, its cell survival rate is 100%, presses for all the other each group: survival rate=(each concentration group light absorption value/matched group light absorption value) × 100% compares with it, mapping.
7.3 result
AEBSF and gabexate mesilate all can suppress the growth of A549 cell and HCC-94 dose dependent, AEBSF half-inhibition concentration is near 100 μ g/mL, and the half-inhibition concentration of gabexate mesilate (is shown in respectively Fig. 3 and Fig. 4) near 700 μ g/mL.
Comparative example 1
The anti-mice implantation test of serpin Antipain
Material
Antipain hydrochloride (purchased from Sigma company) is a kind of reversible serpin separating from microorganism, and the structure of its dihydrochloride is as follows.
ICR mice, age in 8-10 week, purchased from Shanghai western pul-Bi Kai laboratory animal company limited.
Experimental technique
Mice male and female mate, female: hero is to mate at 2: 1.Within second day, female Mus vagina is found cloudy bolt, is decided to be pregnant the 1st day (pregnant d1) same day.
Intrauterine Injection group, in pregnant d4 morning, after anesthesia, is injected uterine cavity from cornua uteri position, every Aconitum carmichaeli Debx. palace subscript enters 0 μ g, 300 μ g, 1000 μ g Antipain medicines (being dissolved in 15 μ l normal saline), injects respectively bilateral uterus.
Result
The number of eggs ovulated (CL) of corpus luteum number representative and the implantation embryo number (embryo) of pregnant 8 days, give at the every side cornua uteri of implantation mice under the antipain treatment conditions of 300 μ g or 1000 μ g dosage, contrast (0 μ g antipain) all less than significantly changing (Fig. 5) with respect to normal saline solution.The difference of corpus luteum number and embryo number represents embryo number that can not implantation, processes between contrast without significant difference (table 5) at antipain.
These results show, compare with gabexate with AEBSF, although antipain belong to equally serpin, the but unrestraint effect to embryo nidation.
Table 5antipain processes the impact of the difference on corpus luteum number and implantation embryo number
| Quadratic sum (sum of square) | Df | Mean square | F | Significance (Si g.) | |
| Between group | 1.063 | 2 | 0.532 | 0.786 | 0.471 |
| In group | 12.175 | 18 | 0.676 | ||
| Add up to | 13.238 | 20 |
List of references:
1.Petruzzelli，G.J.，The biology of tumor invasion，angiogenesis andlymph node metastasi s.ORL J Otorhinolaryngol Relat Spec，2000.62(4)：p.178-85.
2. Ma Fang and Wang Zhibiao, developmental biology problem of tumor. life sciences, 2005.17:p.433-438.
3.Salamonsen，L.A.and G.Nie，Proteases at theendometrial-trophoblast interface：their role in implantation.RevEndocr Metab Disord，2002.3(2)：p.133-43.
4.Sharma，N.，et al.，Substrate specificity determination of mouseimplantation serine proteinase and human kallikrein-related peptidase6by phage display.Biol Chem，2008.
All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (9)
1. a purposes for serpin, described serpin is gabexate or its pharmaceutically acceptable salt, it is characterized in that, described inhibitor is used to compositions or the utensil that preparation suppresses embryo nidation.
2. purposes as claimed in claim 1, is characterized in that, described compositions or utensil are by the compositions of vagina administration or utensil.
3. purposes as claimed in claim 1, is characterized in that, described inhibitor is the gabexate mesilate shown in following formula
4. as the purposes as described in arbitrary in claim 1-3, it is characterized in that, described compositions is pharmaceutical composition.
5. purposes as claimed in claim 4, is characterized in that, described pharmaceutical composition is the compositions that suppresses embryo nidation.
6. purposes as claimed in claim 4, is characterized in that, described pharmaceutical composition is contraceptive; Or described utensil is contraceptive device.
7. purposes as claimed in claim 6, is characterized in that, described contraceptive device comprises pessary or intrauterine device; Or
Described contraceptive comprises vagina gel.
8. one kind is suppressed the utensil of embryo nidation, described utensil is the contraceptive device for women, it is characterized in that, described contraceptive device also comprises that wherein said serpin is gabexate or its pharmaceutically acceptable salt for the releasing parts at vagina or intrauterine release serpin.
9. a purposes for compound, described compound is gabexate or its pharmaceutically acceptable salt, it is characterized in that, described compound is for the preparation of the reversible inhibitor that suppresses embryo nidation.
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| WO2001089449A2 (en) * | 2000-05-25 | 2001-11-29 | Ben Williger | Phospholipase d effectors for therapy and screening |
| US20060293386A1 (en) * | 2005-06-24 | 2006-12-28 | Tsuneo Ozeki | Method for treating cancer |
| CN101259119A (en) * | 2007-03-08 | 2008-09-10 | 上海市计划生育科学研究所 | Application of serine protease inhibitor and its derivative in fertility regulation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2001089449A2 (en) * | 2000-05-25 | 2001-11-29 | Ben Williger | Phospholipase d effectors for therapy and screening |
| US20060293386A1 (en) * | 2005-06-24 | 2006-12-28 | Tsuneo Ozeki | Method for treating cancer |
| CN101259119A (en) * | 2007-03-08 | 2008-09-10 | 上海市计划生育科学研究所 | Application of serine protease inhibitor and its derivative in fertility regulation |
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| Title |
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| 刘宝玉等.合成类丝氨酸蛋白酶抑制剂的研究进展.《中国新药杂志》.2006,第15卷(第5期),334-336. |
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