CN1116075C - Medicine for treating male sterility and weak sperm disease - Google Patents
Medicine for treating male sterility and weak sperm disease Download PDFInfo
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- CN1116075C CN1116075C CN 99121933 CN99121933A CN1116075C CN 1116075 C CN1116075 C CN 1116075C CN 99121933 CN99121933 CN 99121933 CN 99121933 A CN99121933 A CN 99121933A CN 1116075 C CN1116075 C CN 1116075C
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Abstract
The present invention relates to a medicament, namely urokinase for treating male sterility and weak sperm diseases, an urokinase preparation and the new application of the urokinase preparation for treating male sterility and weak sperm diseases. In the present invention, an injection and an oral preparation can be prepared by a traditional preparation formula. In the present invention, beta-cyclodextrin is used for covering the urokinase, and then the compound is prepared into a buccal medical tablet with physiological activity by a distinct preparation technology so as to enhance the curative effects of the urokinase, reduce the side effects of the urokinase and enhance stability. Experiment results prove that the medicament keeps the activity of urokinase and effectively treats male sterility and weak sperm diseases.
Description
What the present invention relates to is a kind of treatment male sterility, azoospermia medicine---urokinase, and specifically, what the present invention relates to is the method and the purposes aspect treatment male azoospermia, male sterility obstacle thereof of urokinase medicine, this medicine of preparation.
The beginning of the fifties, Williams finds urokinase at first in human urine, the latter stage seventies, people such as Propping and Asted isolated two kinds of former activity factors of lyase (uPA) from seminal fluid, and with having uPA in biochemical method confirmer's sperm, it is similar urokinase on immunology and biological activity.Researcheres were produced uPA multi-resistance and monoclonal antibody with the urokinase immune animal afterwards, were used for further studying uPA, further discovered, in fact urokinase is exactly present uPA in the human body of early discovery.From in human urine, finding urokinase in early days and from seminal fluid, isolating two kinds of former activity factors of lyase (uPA) so far, in the prior art, urokinase (Urokinase) is mainly used in the treatment thrombosis, route of administration is injection, see state-promulgated pharmacopoeia, never the people links together urokinase with the quality of improving sperm.
The inventor finds that in the new drug research process urokinase (Urokinase) has the new purposes of treatment male sterility obstacle, azoospermia, does not still have report and use at present both at home and abroad.
The object of the present invention is to provide a kind of medicine---the urokinase that can effectively treat male sterility obstacle, azoospermia.
Another object of the present invention provides the prescription and the preparation technology of multiple preparation urokinase preparation, comprises oral buccal tablet, oral capsule, injection and suppository.
Another object of the present invention is the relation of the activity of urokinase in research sperm and the refining with male sterility obstacle and azoospermia, thereby provides a kind of activity by urokinase in check sperm and the refining to determine the method for male sterility obstacle and azoospermia.
A further object of the present invention is that urokinase is applied to treat male sterility obstacle and azoospermia.
In order to finish above-mentioned task, the technical solution used in the present invention is:
At first study urokinase type plasminogen activator uPA and urokinase type plasminogen activator receptor uPAR in people's sperm and the refining, and the variation of uPA enzymatic activity and content in azoospermia patient sperm and the refining.Urokinase type plasminogen activator (uPA) is a kind of serine proteinase enzyme, and it changes plasminogen into fibrinolysin; Urokinase type plasminogen activator receptor (uPAR) is that a kind of molecular weight is 55-66,000 strand glycoprotein, and it is attached on the plasma membrane by sugared phosphinositides, and the physiological action of uPA is not clear fully as yet in human seminal plasma and the sperm.
In addition, the present invention mainly studies from the following aspects:
1. observe content, the activity of urokinase in the biologic activity of the content of urokinase in normal man's refining and enzyme and the sperm and distribute.
2. observe asthenospermia, urokinase content and biologic activity change in conventional all other basic normal infertility man refinings of seminal fluid and the sperm.
3. to asthenospermia, use urokinase in the same sterile man's body of other condition after, observe that motility of sperm changes in the seminal fluid.
4. to after adding urokinase in the stripped seminal fluid of above-mentioned condition patient, observe motility of sperm and change.
Specifically, the present invention has mainly adopted immunohistochemical method, and uPA in people's sperm and uPA receptor are positioned observation.With agarose-fibrin-flat band method+immune impedance the activity of uPA in people's sperm and the refining is measured, the content of uPA in people's sperm and the refining is measured with ELISA-double antibodies sandwich method.In addition to sterile man of azoospermia and the normal man of seminal parameters, external add uPA after, in the time of 30,60,90 and 120 minutes, light microscopic is observed the change of motility of sperm down:
1. the SABC method comprises immune colloid gold colored light sem observation and immune colloid gold dyeing (monoclonal antibody swell antibody+polyclonal antibody double label method), does not redye, and carries out electron microscopic observation, set up contrast dyeing to compare observation simultaneously, this method high specificity, highly sensitive, experimental result is satisfied.
2. agarose-fibrin-flat band method+immune impedance: by measuring the size of dissolving circle, understand the enzymatic activity of uPA, the diameter of the transparent circle of appearance and the logarithm of uPA are linear, understand the activity of enzyme by the content of uPA, this method is accurate, and the energy direct reaction goes out the activity of enzyme.
3.ELISA-the double antibodies sandwich method adopts the double antibodies sandwich method to carry out the uPA quantitative assay, method is highly sensitive, high specificity, similar to the radioimmunity measured result, its correlation coefficient of standard curve of surveying with this method is 0.999, and is little with the variation within batch coefficient between batch, is respectively 10.46% and 2.26%.
Laboratory observation is the result show:
1. uPA mainly is distributed on interior adventitia of perforatorium and the head serous coat in normal man's sperm, the unexpected essence of finding
Also there is uPA in the position that sub-afterbody stage casing mitochondrion is concentrated.All can find uPAR at the position that uPA occurs.
2. uPA activity and content significantly are lower than normal man's group in the azoospermia patient sperm, the active and content branch of uPA
Be not obvious positive correlation with motility of sperm and motility rate.
3. external urokinase stimulation test shows, add urokinase after, the work of sperm in the time of 30,60,90,120 minutes
Rate is improved, and the number of propulsion sperm also has increase.Relatively, urokinase is bright before and after the vivo medicine-feeding
Show average motility rate of sperm and the propulsion ability of improving.
4. azoospermia and the seminal fluid bad person of liquefying, the active and content of uPA significantly is lower than normal man's group in the refining, unites
Upward there were significant differences in meter.
According to above-mentioned experimental result, reduce following some:
1. uPA may combine with uPAR and just bring into play physiological action.Owing to have the uPA receptor in the sperm, no
Getting rid of uPA is that sperm combination in the reproductive tract running is got on.
2. the existence of sperm tail stage casing uPA and uPAR has special physiological significance, it and sperm head uPA
Function may be different.This position is the place that mitochondrion is concentrated, supposition may with energy utilization and sperm
Motion relevant.
3. the vigor of uPA and sperm and motility rate are proportionate.
4. uPA may participate in the liquefaction of seminal fluid and the viscosity of reduction seminal fluid, helps sperm and does propulsion.
5. uPA might develop into a kind of medicine for the treatment of the azoospermia patient.
6. further study uPA, might reach the effect of male-contraception by suppressing the uPA activity.
In sum, we sum up marked feature of the present invention and are:
1, adopts the two mark of gold colloidal immunoelectron microscopic method, on form, intuitively, accurately confirmed uPA first at home and abroad
With the distribution of uPAR in people's sperm.
2, propose the sperm tail stage casing both at home and abroad first and have uPA and uPAR, and confirmed from morphology,
Infer possible physiological significance simultaneously.
3, propose in azoospermia patient and the sperm uPA content both at home and abroad first and reduce relevantly, propose uPA with active
Can be used as a kind of medicine for the treatment of azoospermia.
4, the viscosity that uPA in the refining may participate in the liquefaction of seminal fluid and reduce seminal fluid is proposed, before helping sperm and doing
To motion.
Because uPA has the vigor of raising sperm and the effect of motility rate, and can participate in the liquefaction of seminal fluid and the viscosity of reduction seminal fluid, help sperm and do propulsion.Therefore, uPA can become a kind of new drug for the treatment of male sterility obstacle and azoospermia.
A kind of medicine for the treatment of male sterility obstacle and azoospermia, main component is a urokinase in this medicine, it is characterized in that containing in this medicine the urokinase of 10000-100000 unit, it is as follows to fill a prescription:
Amounts of components
Urokinase 10000-100000 unit
Beta-schardinger dextrin-0-60mg
Mannitol 0-100mg
Acid medium 0-20mg
Polyethylene Glycol 0-20mg
Binding agent 0-25mg
Starch 0-150mg
Cane sugar powder 0-250mg
Magnesium stearate 0-6mg
Carboxymethyl starch sodium 0-22mg
Tween 0-2mg
Semi-synthetic fatty acid enzyme 0-40mg
Amount to 10000-100000 unit to 300mg
The medicine of treatment male sterility obstacle of the present invention and azoospermia, the consumption that it is characterized in that described urokinase is preferably between 12000-80000 unit.
The medicine of treatment male sterility obstacle of the present invention and azoospermia when the content of inclusion agents, adhesive and other adjuvant is zero, adds normal saline in the urokinase, can make injection.
Use urokinase treatment male sterility obstacle and azoospermia, can be with traditional injection system administration, but with the injection system administration, very inconvenient for user on the one hand, be difficult to overcome the side effect of urokinase itself on the other hand; During oral administration, because enzyme may make the urokinase inactivation and influences the due effect of urokinase the effect of urokinase in the stomach.For both convenient uses, guarantee the activity of urokinase again simultaneously, the present invention utilizes the characteristic of beta-schardinger dextrin-molecular microcapsule, adopts the beta-cyclodextrin inclusion compound urokinase.Because the beta-schardinger dextrin-molecule is arranged in the form of a ring; hydroxyl on the glycoside base all is positioned at the periphery of circulus; so the periphery is a hydrophilic; inside is hydrophobicity; form cylindric; it is protected that urokinase can enter cyclodextrin inside, also can the hydrogen bond type of attachment strengthen its stability, can guarantee the stability of urokinase.
The prescription of urokinase buccal tablet of the present invention is as follows:
Amounts of components
Urokinase 10000-100000 unit
Beta-schardinger dextrin-6-60mg
Mannitol 30-100mg
Stearic acid 2-20mg
Polyethylene Glycol 2-20mg
Cellulose 1.5-15mg
Cane sugar powder 5-250mg
Magnesium stearate 1-6mg
Adjuvant is some an amount of
Add up to 300mg
Urokinase buccal tablet of the present invention, the consumption that it is characterized in that described urokinase is preferably between 12000-80000 unit.
Oral or capsular prescription are:
The prescription consumption
Urokinase 10000-100000 unit
Beta-schardinger dextrin-0-60mg
Starch 30-150mg
Cellulose 5-25mg
Carboxymethyl starch sodium 0-60mg
Magnesium stearate 0-6mg
Silicon dioxide 0-10mg
Cyclodextrin in the present invention prescription can be selected from beta-schardinger dextrin-and multiple derivant thereof, for example HP-(HP-β-CD), ethyl-beta-schardinger dextrin-(E-β-CD), DM-(DM-β-CD), TM-(TM-β-CD), G 1-CD (G
1β-CD), G 2-CD (G
2β-CD), G 3-(G
3β-CD), 2G1-(2G
1β-CD), 2G2-(2G
2β-CD) etc.Require further slow release as desire, ((TE-β-CD) etc. also can be two or more mixture for DE-β-CD), triethyl group-beta-schardinger dextrin-also can to select DE-; Preferred DM-, ethyl-beta-schardinger dextrin-(E-β-CD) and triethyl group-beta-schardinger dextrin-.
Acid medium in above-mentioned prescription can select to use stearic acid, palmitic acid, Palmic acid, ascorbic acid or the like.Preferred stearic acid, ascorbic acid and Palmic acid.
Binding agent in above-mentioned prescription is optional from following a kind of or combination: hypromellose, ethylmethylcellulose, ethyl cellulose, poly-a-hydroxyl second methacrylate, polyvidone, polyvinyl acetate, cellulose acetate, dextrin, from dextrin, microcrystalline Cellulose, methylcellulose, acrylic acid methyl ester., acrylic resin I-IV number, hydroxyethylmethyl-cellulose, hydroxyethyl-cellulose, card pool nurse, sodium carboxymethyl cellulose, carboxymethylcellulose calcium, low-substituted hydroxypropyl cellulose, starch or the like, the stability of so further enhancing urokinase.Preferred adhesive is selected from hypromellose, ethylmethylcellulose and ethyl cellulose.
Polyethylene Glycol in the above-mentioned prescription also can be with the Polyethylene Glycol of other various models, as Macrogol 4000, and Polyethylene Glycol 7000, Polyethylene Glycol 8000 also can be the derivant of Polyethylene Glycol or be fit to other polymer of the present invention.
Above-mentioned prescription of the present invention can be selected different binding agents, different acid mediums, different cyclodextrin and derivant and different polymer.
Above-mentioned prescription can be made oral tablet, oral capsule, granule, suppository and be fit to other preparations of the present invention.
In addition, as long as reach identical blood drug level, be used for urokinase of the present invention and also can make injection, oral capsule, oral tablet, granule, suppository and be fit to other preparation of the present invention with traditional method.
Above-mentioned adjuvant, consumption and dosage form can have multiple variation, and this is conspicuous for a person skilled in the art, differ at this and one list.But should be understood that all reach the variation of dosage form in above-mentioned formula range, all should be included in the scope of the present invention.
The preparation technology of urokinase buccal tablet of the present invention is as follows:
1. earlier polyethylene glycol 6000 is dissolved with warm water, cooling adds urokinase and stirs, and adds beta-schardinger dextrin-and constantly stirs, and it is complete that about 2--6hr makes it enclose, after the filtration, with the clean clathrate that promptly gets of clear water.
2. with mannitol, stearic acid, cane sugar powder mix homogeneously, add clathrate, fully add 3% hydroxypropyl emthylcellulose behind the mix homogeneously again, make soft material, then with 16 mesh sieve system granules, with 40--70 ℃ of forced air drying 4--8hr, with 16 mesh sieves arrangement granule.
3. the granule of putting in order is added magnesium stearate, mix homogeneously gets final product; Use the particular manufacturing craft tabletting, sheet heavily is about the 300mg/ sheet.
Oral tablet of the present invention, capsule, particulate preparation technology are as follows:
1. after HPMC being added an amount of EtOH dispersion, add 60-80 ℃ of distilled water diluting, after airtight placement is spent the night, cross 70-80 mesh sieve standby (grinding agent, binding agent) to needs concentration.
2. all adjuvants are added, stirring is pasty state, adds urokinase pharmacy soft material.
3. soft material is granulated 40-60 ℃ of aeration-drying, 18 order granulate with 18 orders.
4. add carboxymethyl starch sodium, magnesium stearate mix homogeneously tabletting behind the dry granulate or add silicon dioxide glue capsule.
When preparing oral tablet or capsule with beta-cyclodextrin inclusion compound, preparation technology is with the preparation process of buccal tablet, and different is the technology of last molding.
The present invention is in the male genetic field, and------urokinase has been filled up domestic and international blank to the research of uPA and uPAR and motility of sperm and the treatment male sterility obstacle that proposes thus and the medicine of azoospermia; The present invention is on the basis of carrying out galenic pharmacy research, adopt beta-schardinger dextrin-that urokinase is carried out enclose, add other supplementary materials, and cooperate unique preparation process, be developed into a kind of medicine for the treatment of male sterility obstacle, azoospermia, being characterized as of medicine: the tablet that physiologically active is arranged, suck type, the defective that urokinase itself is existed is overcome, and makes the raising evident in efficacy of urokinase, and the side effect that has alleviated this material, strengthened stability; Can bring into play the new purposes of its treatment male sterility obstacle, azoospermia effectively.
Because uPA and motility of sperm are closely related, the activity of urokinase is to part azoospermia (low sperm activity) or seminal fluid liquefy bad the have clear and definite cause of disease and diagnostic effect in check sperm and the refining, and this method is simple, accurately, reliably, special instruments and equipment can not carried out in basic hospital clinical laboratory.
Experimental result shows that this medicine has kept the activity of urokinase, can bring into play the new purposes of its treatment male sterility obstacle, azoospermia effectively.The cause of disease that causes low sperm activity is more, and it may be one of cause of disease that urokinase activity reduces, and the activity of therefore analyzing urokinase in sperm and the refining has the certain significance to the treatment of instructing azoospermia.To part low sperm activity or seminal fluid abnormal liquefaction, urokinase has good therapeutic effect, and good promotion prospect is arranged.
Embodiment one
Prescription of the present invention and preparation technology's key is the enclose of beta-schardinger dextrin-to the principal agent composition.In the present embodiment, clathrate select DM-(DM-β-CD), its prescription is as follows:
Amounts of components
Urokinase 20000 units
DM-10mg
Mannitol 46mg
Stearic acid 6mg
Hydroxypropyl emthylcellulose 30mg
Polyethylene glycol 6000 3mg
Magnesium stearate 10mg
Cane sugar powder (not sulfur-bearing) 220mg
Add up to every heavy 300mg
The preparation technology of the described urokinase buccal tablet of present embodiment is as follows:
1. earlier polyethylene glycol 6000 is dissolved with warm water, cooling adds urokinase and stirs, and adds beta-schardinger dextrin-and constantly stirs, and it is complete that about 2hr makes it enclose, after the filtration, with the clean clathrate that promptly gets of clear water.
2. with mannitol, stearic acid, cane sugar powder mix homogeneously, add clathrate, fully add 3% hydroxypropyl emthylcellulose behind the mix homogeneously again, make soft material, then with 16 mesh sieve system granules, with 40 ℃ of forced air drying 8hr, with 16 mesh sieves arrangement granule.
3. the granule of putting in order is added magnesium stearate, mix homogeneously gets final product.Use the particular manufacturing craft tabletting, sheet heavily is the 300mg/ sheet.
Embodiment two
In the present embodiment, clathrate is selected ethyl-beta-schardinger dextrin-, and its prescription is as follows:
Amounts of components
Urokinase 10000 units
Ethyl-beta-schardinger dextrin-60mg
Mannitol 100mg
Palmitic acid 17.5mg
Polyethylene glycol 6000 20mg
Ethylmethylcellulose 1.5mg
Cane sugar powder (not sulfur-bearing) 100mg
Magnesium stearate 1mg
Add up to every heavy 300mg
The preparation technology of the described urokinase buccal tablet of present embodiment is as follows:
1. earlier polyethylene glycol 6000 is dissolved with warm water, cooling adds urokinase and stirs, and adds beta-schardinger dextrin-and constantly stirs, and it is complete that about 6hr makes it enclose, after the filtration, with the clean clathrate that promptly gets of clear water.
2. with mannitol, palmitic acid, cane sugar powder mix homogeneously, add clathrate, fully add 3% ethylmethylcellulose behind the mix homogeneously again, make soft material, then with 16 mesh sieve system granules, with 75 ℃ of forced air drying 2hr, with 16 mesh sieves arrangement granule.
3. the granule of putting in order is added magnesium stearate, mix homogeneously gets final product.Use the particular manufacturing craft tabletting, sheet heavily is the 300mg/ sheet.
Embodiment three
Prescription:
Amounts of components
Urokinase 100000 units
TM-6mg
Mannitol 30mg
Ascorbic acid 10mg
Macrogol 4000 15mg
Ethyl cellulose 1.5mg
Cane sugar powder (not sulfur-bearing) 210mg
Magnesium stearate 6mg
Add up to every heavy 300mg
The preparation technology of the described urokinase buccal tablet of present embodiment is as follows:
1. earlier Macrogol 4000 is dissolved with warm water, cooling adds urokinase and stirs, and adds beta-schardinger dextrin-and constantly stirs, and it is complete that about 4hr makes it enclose, after the filtration, with the clean clathrate that promptly gets of clear water.
2. with mannitol, ascorbic acid, cane sugar powder mix homogeneously, add clathrate, fully add 3% ethyl cellulose behind the mix homogeneously again, make soft material, then with 16 mesh sieve system granules, with 55 ℃ of forced air drying 4hr, with 16 mesh sieves arrangement granule.
3. the granule of putting in order is added magnesium stearate, mix homogeneously gets final product.Use the particular manufacturing craft tabletting, sheet heavily is the 300mg/ sheet.
Embodiment four
Oral tablet prescription and preparation technology:
Amounts of components
Urokinase 20000 units
TM-11mg
Starch 150mg
Low-substituted hydroxypropyl cellulose (L-HPC) 20mg
HPMC 3% concentration carboxymethyl starch sodium (CMS-Na) 11mg magnesium stearate (M, S) 3mg
Wherein, ethanol and distilled water can mix with arbitrary proportion.
The preparation technology of the described urokinase oral tablet of present embodiment is as follows:
1. HPMC is added an amount of ethanol and disperse, add 80 ℃ of distilled water dilutings then to needs concentration, airtight placement is spent the night, and crosses 80 mesh sieves standby (grinding agent, binding agent).
2. beta-schardinger dextrin-is added the second alcohol and water, HPMC three's mixed liquor, stirring is pasty state, adds urokinase, it is standby to grind enclose 40 minutes.
3. with starch, L-HPC mix homogeneously, add the good principal agent of enclose, stir, add an amount of binding agent and make soft material.
4. soft material is granulated 50 ℃ of aeration-dryings, 16 order granulate with 16 orders.
5. add carboxymethyl starch sodium behind the dry granulate, the magnesium stearate mix homogeneously can tabletting.
Embodiment five
Oral tablet prescription and preparation technology:
Amounts of components
Urokinase 70000 units
G 1-CD 60mg
Starch 100mg
Low-substituted hydroxypropyl cellulose (L-HPC) 2.5mg
Hydroxypropyl emthylcellulose (HPMC) 2.5mg-
Ethanol (EtOH) HPMC 3% concentration
Distilled water-
Carboxymethyl starch sodium (CMS-Na) 26mg
Magnesium stearate (M, S) 2mg
Wherein, ethanol and distilled water can mix with arbitrary proportion.
1. after HPMC being added an amount of EtOH dispersion, add 80 ℃ of distilled water dilutings, after airtight placement is spent the night, cross 80 mesh sieves standby (grinding agent, binding agent) to needs concentration.
2. all adjuvants are added, stirring is pasty state, adds urokinase pharmacy soft material.
3. soft material is granulated 50 ℃ of aeration-dryings, 18 order granulate with 18 orders.
4. add carboxymethyl starch sodium, magnesium stearate mix homogeneously tabletting behind the dry granulate.
Embodiment six
Capsule prescription and preparation technology:
Amounts of components
The β of urokinase 20000 units-CD 10mg starch 100mgL-HPC 10mg
HPMC 3% concentration SiO
25mg
The preparation technology of the described urokinase oral capsule of present embodiment is as follows:
1. after HPMC being added an amount of EtOH dispersion, add 60 ℃ of distilled water dilutings, after airtight placement is spent the night, cross 70 mesh sieves standby (grinding agent, binding agent) to needs concentration.
2. β-CD is added 1 (HPMC liquid) and stir and be pasty state, add urokinase, it is standby to grind enclose 460 minutes.
3. with starch, L-HPC mix homogeneously, good principal agent stirs to add enclose, adds suitable amount of adhesive 1 pharmacy soft material.
4. soft material is granulated 50 ℃ of aeration-dryings, 18 order granulate with 18 orders.
5. add silicon dioxide behind the dry granulate, mixing fill 2
#Capsule.
Embodiment seven
Capsule prescription and preparation technology: the amounts of components urokinase 20000 starch 110mgL-HPC 10mg of unit
HPMC 3% concentration SiO
25mg
The preparation technology of the described urokinase oral capsule of present embodiment is as follows:
Step 1-3 is with embodiment five, and step 4 is:
4. add silicon dioxide behind the above-mentioned dry materials granulate, mixing fill 2
#Capsule.
Embodiment eight
Suppository prescription and preparation technology:
Amounts of components
Urokinase 20000 units
β-CD 10mg
Tween 80 2mg
The semi-synthetic fatty acid enzyme 40mg of 36 types
The preparation technology of the described urokinase suppository of present embodiment is as follows:
1. β-CD is added an amount of EtOH and stir and be pasty state, adding urokinase, to grind enclose 40 minutes standby.
2. with 38 ℃ of insulations after the semi-synthetic fatty acid enzyme heating for dissolving of 36 types, add the clathrate of principal agent, fully mix homogeneously insulation.
3. mix homogeneously, insulation thing are irritated the film cooling forming.
Embodiment nine
Urokinase injection liquid: other component in the prescription is 0, gets urokinase dry powder 40000 units, during use, adds normal saline in proportion, promptly becomes urokinase injection liquid.The Position Research of uPA (uPA) and urokinase type fibrinolysin unit's activity factor receptor (uPAR) in embodiment ten people's sperms.
The latter stage seventies human biochemistry such as Propping and Asted method confirmer's sperm in have uPA (uPA).Thereafter, more scholar has carried out the follow up study to the physiological significance that it exists, yet the position that uPA exists in sperm is short in understanding.In early days the Biochemical Research to uPA adopts the fibrin plate method more, because of this method is subjected to the interference of other serine proteinase enzyme in the seminal fluid, lacks specificity.Although the eighties and the nineties adopt enzyme linked immunosorbent assay and radioimmunoassay to measure, and have confirmed the existence of uPA, the position that uPA is existed in sperm is a kind of supposition always, lacks the evidence of morphology aspect.In addition also there is arguement in the source of uPA in the sperm always, have the scholar infer uPA be sperm by the process of reproductive tract in conjunction with getting on.If, at first must prove the receptor that has uPA on the sperm membrane,, all there is not report both at home and abroad to whether there being the document of uPA receptor in the sperm in conjunction with what get on.Also have a kind of uPA of thinking performance physiological action by with the uPA receptors bind after just show physiological effect, shown the importance of uPAR existence.Therefore will further understand uPA, the Position Research of carrying out uPA and uPAR is significant.
We successively adopt immunohistochemical method, comprise that immune colloid gold light microscopic method and gold colloidal double labelling electron microscopy, SABC fluorescence method etc. study the outer sperm of normal man's emitting body.The first antibody of effect is respectively from domestic BIO ENGINEERING INST MILITARY and Denmark Finsen laboratory.Experimental result shows that uPA and uPAR mainly are distributed on interior adventitia of perforatorium and the head serous coat.What is interesting is that we also find to have in the sperm tail stage casing distribution of uPA and uPAR, this position is the place that mitochondria of sperms is concentrated, and mitochondrion is mainly sperm motion institute energy requirement is provided, and more than finds to be domestic and international reported first.According to the position of uPA and uPAR distribution, infer that their physiological action may be different.The uPA and the uPAR that are positioned at the perforatorium position may participate in acrosome reaction, help sperm that the identification and the sperm of ovum are passed zona pellucida and cervical mucus; The uPA that is positioned at sperm tail may be relevant with sperm motility with uPAR, do not adhere to each other with tube wall and sperm in epididymis to guarantee sperm, and the liquefaction to seminal fluid simultaneously has facilitation.The content of uPA and relation active and sperm motility in embodiment 11 normal men and the azoospermia man sperm.
After people's seminal fluid emitting body is outer, liquefaction in about 5-30 minute, the sperm after the liquefaction has motor capacity, comprises the original place fine motion, slowly propulsion, middling speed motion and rapid movement, some sperm inertia.It may be that Necrospermia also can be the sperm of living that common light microscopic is observed down this part motionless sperm, former thereby can not move because of certain.By the percentage rate of movable and spermatium being represented the motility rate of sperm.The sperm of all motions is made the speed of propulsion and the quality that percent is judged motility of sperm according to them.This research is exactly the relation of understanding uPA and sperm motility rate and propulsion ability.Exist uPA and uPAR to be proved in front in people's sperm, but less about the uPA content in normal man and the azoospermia man sperm with active report, by analysis, can recognize the influence of uPA to sperm motility to uPA in the azoospermia man sperm.
This research contrasts the line correlation analysis of going forward side by side to uPA content and activity in infertility man's sperm of azoospermia (the low and propulsion ability of sperm motility rate) simultaneously by to normal man uPA content and active mensuration.Agarose-fibrin plate method binding immunoassay blocked method is adopted in the uPA determination of activity.Assay adopts ELISA double antibodies sandwich method.Experimental result is found uPA content and the active content that significantly is lower than uPA in the normal fertility man sperm in the azoospermia man sperm, and utmost point significance meaning is arranged between two groups, finds that through correlation analysis the content of uPA and sperm motility rate and vigor have obvious dependency.We find that also the uPA activity also is higher than the sterile man's group of azoospermia in normal man's sperm, and statistical procedures has utmost point significant difference.Sperm uPA activity value and sperm motility rate, vigor are also linearly relevant, and statistical significance is arranged.According to above-mentioned experimental result, we think that motility rate and the propulsion of uPA and sperm has certain relation in the sperm.Because uPA may participate in many aspects of reproductive process, as the penetrating of the liquefaction of the maturation of sperm, motion, seminal fluid, cervical mucus, with the combining of ovum, acrosome reaction, the growth that penetrates zona pellucida, embryo and implantation etc.It is sterile that the azoospermia man causes, may be uPA content with active low and cause the sperm motility ability, can not run to due to women's ampulla of uterine tube becomes pregnant, and causes sterile but can not get rid of fully because of uPA lowly can not participate in other reproductive process.The motion that shows uPA and sperm in a word has dependency relation.Embodiment 12 uPA are to the experimentation of azoospermia patient motility of sperm influence
The beginning of the fifties, Williams found urokinase at first in human urine, and the latter stage seventies, people such as Propping isolated two kinds of former activity factors of lyase, similar urokinase on immunology and biological activity from seminal fluid.Researcheres were produced uPA multi-resistance and monoclonal antibody with the urokinase immune animal afterwards, were used for further studying uPA, and in fact urokinase is exactly present uPA in the early discovery human body.By after adding urokinase in normal healthy volunteer's seminal fluid, observing, find that urokinase has the effect that improves sperm motility, confirm the effect that improves sperm motility rate and propulsion ability that truly has of uPA.We have observed 28 routine azoospermia men.Experimental technique is the external test tube method of seminal fluid that exsomatizes, with twice semen analysis seminal parameters remove motility rate and or low other all normal specimen of vigor as object of study, treat to add respectively after seminal fluid liquefies the urokinase of variable concentrations, observe to add behind the urokinase 30, the variation of sperm motility rate in the time of 60,90 and 120 minutes.Another experiment is to giving urokinase in the azoospermia man body, use 10 days continuously, observing the variation with sperm motility rate before and after the urokinase and propulsion ability.Learn to handle by statistics, when experimental result is found to add behind the urokinase 30,60,90 and 120 minutes sperm motility rate is all increased, the number of propulsion sperm also has increase.After giving urokinase in the body, the average motility rate of sperm rises to 64.71% by 44.64%, and utmost point significant difference is arranged.Experiment in vivo and vitro proof urokinase has the effect that improves sperm motility rate and propulsion ability.This effect may be relevant with reduction seminal fluid viscosity, but do not get rid of the possibility that might improve the sperm motility ability by other mechanism.The relation of uPA activity and content and motility of sperm in embodiment 13 refinings
People such as MacGregor find to exceed 60-100 in the concentration ratio blood of plasminogen activating factors in the refining doubly.The physiological action that uPA exists in the refining is not very clear, and supposition may be relevant with the liquefaction of seminal fluid.The good seminal fluid that liquefies helps sperm and does propulsion.The bad seminal fluid that liquefies, can see in the refining elongated fibrin and mutually net knit the sperm motility space reduced, the motion of sperm is restricted.We have measured the 20 routine seminal fluid uPA activity in bad man's refining that liquefies, and simultaneously with the normal man of 15 examples in the refining uPA activity compare, found that urokinase activity is starkly lower than normal man's group in bad group of refining of liquefaction, utmost point significant difference is arranged.We also carry out urokinase to the infertility man of low group of 22 routine sperm motility rates and aspermia liquefaction bad phenomenon and measure in addition, found that the content of urokinase in the refining is lower than the content of seminal parameters normal group man refining urokinase, has utmost point significant difference.According to aforesaid research, low (azoospermia) patient of sperm motility rate, uPA is active in the sperm obviously reduces with content, discovers that originally uPA content also shows low value simultaneously in the azoospermia patient refining.Physiological significance about uPA in the refining or title urokinase removes reduction seminal fluid viscosity, outside the favourable sperm motility, whether also has the effect of others, is still waiting further to study.
Claims (11)
1, a kind of medicine for the treatment of male sterility obstacle, azoospermia---main component is a urokinase, it is characterized in that containing in this medicine the urokinase of 10000-100000 unit, and it is as follows to fill a prescription:
Amounts of components
Urokinase 10000-100000 unit
Beta-schardinger dextrin-0-60mg
Mannitol 0-100mg
Acid medium 0-20mg
Polyethylene Glycol 0-20mg
Binding agent 0-25mg
Starch 0-150mg
Cane sugar powder 0-250mg
Magnesium stearate 0-6mg
Carboxymethyl starch sodium 0-22mg
Tween 0-2mg
Semi-synthetic fatty acid enzyme 0-40mg
Amount to 10000-100000 unit to 300mg.
2, the medicine of treatment male sterility obstacle according to claim 1, azoospermia, the consumption that it is characterized in that urokinase is between 12000-80000 unit.
3, the medicine of treatment male sterility obstacle according to claim 1, azoospermia is characterized in that other component is 0 in the medicine, when the urokinase consumption is 10000-100000 unit, adds an amount of normal saline, makes injection.
4, the medicine of treatment male sterility obstacle according to claim 1, azoospermia is characterized in that the prescription of buccal tablet is:
The prescription consumption
Urokinase 10000-100000 unit
Beta-schardinger dextrin-6-60mg
Mannitol 30-100mg
Stearic acid 2-20mg
Polyethylene glycol 6000 2-20mg
Hydroxypropyl emthylcellulose 1.5-15mg
Cane sugar powder 5-250mg
Magnesium stearate 1-6mg
Add up to every heavy 300mg
5, the medicine of treatment male sterility obstacle according to claim 1, azoospermia is characterized in that oral or capsular prescription are:
The prescription consumption
Urokinase 10000-100000 unit
Beta-schardinger dextrin-0-60mg
Starch 30-150mg
Cellulose 5-25mg
Carboxymethyl starch sodium 0-60mg
Magnesium stearate 0-6mg
Silicon dioxide 0-10mg
6, the medicine of treatment male sterility obstacle according to claim 1, azoospermia is characterized in that described beta-schardinger dextrin-is HP-(HP-β-CD), ethyl-beta-schardinger dextrin-(E-β-CD), DM-(DM-β-CD), TM-(TM-β-CD), G 1-CD (G
1β-CD), G 2-CD (G
2β-CD), G 3-(G
3β-CD), 2G1-(2G
1β-CD), 2G2-(2G
2β-CD), require further slow release as desire also can select DE-(DE-β-CD), triethyl group-beta-schardinger dextrin-(TE-β-CD), also can be two or more mixture.
7, according to the treatment male sterility obstacle of claim 6, the medicine of azoospermia, wherein said beta-schardinger dextrin-is selected from DM-, ethyl-beta-schardinger dextrin-(E-β-CD) and triethyl group-beta-schardinger dextrin-.
8, treatment male sterility obstacle according to claim 1, the medicine of azoospermia is characterized in that binding agent wherein is selected from the mixture of following one or more: hydroxypropyl emthylcellulose, ethylmethylcellulose, ethyl cellulose, poly-a-hydroxyethyl meth acrylate, polyvidone, polyvinyl acetate, cellulose acetate, dextrin, white dextrin, microcrystalline Cellulose, methylcellulose, acrylic acid methyl ester., acrylic resin I-VI number, hydroxyethylmethyl-cellulose, hydroxyethyl-cellulose, card pool nurse, sodium carboxymethyl cellulose, carboxymethylcellulose calcium.
9, the medicine of treatment male sterility obstacle according to claim 1, azoospermia, it is characterized in that the Polyethylene Glycol in the described prescription, can be Macrogol 4000, polyethylene glycol 6000, the Polyethylene Glycol of Polyethylene Glycol 7000 or other various models or the derivant of Polyethylene Glycol.
10, a kind of medicine for the treatment of male sterility obstacle, azoospermia---the preparation technology of urokinase buccal tablet is characterized in that:
1. earlier Polyethylene Glycol is dissolved with warm water, cooling adds urokinase and stirs, and adds beta-schardinger dextrin-and constantly stirs, and it is complete to make it enclose after 2-6 hour, filters, and cleans with clear water and promptly gets clathrate;
2. with mannitol, stearic acid, cane sugar powder adjuvant mix homogeneously, add clathrate, fully add 3% hydroxypropyl emthylcellulose behind the mix homogeneously again, make soft material,,, put in order with 16 mesh sieves with 40-70 ℃ of forced air drying 4-8 hour then with 16 mesh sieve system granules;
3. the granule of putting in order is added magnesium stearate, mix homogeneously gets final product; Use the particular manufacturing craft tabletting, sheet heavily is the 300mg/ sheet.
11, a kind of medicine for the treatment of male sterility obstacle, azoospermia---oral of urokinase or capsular preparation technology is characterized in that:
1. after HPMC being added an amount of E+OH dispersion, add 60-80 ℃ of distilled water diluting, after airtight placement is spent the night, cross 70-80 mesh sieve standby (grinding agent, binding agent) to needs concentration;
2. all adjuvants are added, stirring is pasty state, adds urokinase pharmacy soft material;
3. soft material is granulated 40-60 ℃ of aeration-drying, 18 order granulate with 18 orders;
4. add carboxymethyl starch sodium, magnesium stearate mix homogeneously tabletting behind the dry granulate or add silicon dioxide glue capsule.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99121933 CN1116075C (en) | 1998-10-16 | 1999-10-15 | Medicine for treating male sterility and weak sperm disease |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98120517.8 | 1998-10-16 | ||
| CN98120517 | 1998-10-16 | ||
| CN 99121933 CN1116075C (en) | 1998-10-16 | 1999-10-15 | Medicine for treating male sterility and weak sperm disease |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1260997A CN1260997A (en) | 2000-07-26 |
| CN1116075C true CN1116075C (en) | 2003-07-30 |
Family
ID=25744759
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 99121933 Expired - Fee Related CN1116075C (en) | 1998-10-16 | 1999-10-15 | Medicine for treating male sterility and weak sperm disease |
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| Country | Link |
|---|---|
| CN (1) | CN1116075C (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102653753A (en) * | 2012-05-17 | 2012-09-05 | 江南大学 | Method for suppressing soybean lipoxygenase activity by use of cyclodextrin |
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1999
- 1999-10-15 CN CN 99121933 patent/CN1116075C/en not_active Expired - Fee Related
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| Publication number | Publication date |
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| CN1260997A (en) | 2000-07-26 |
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