CN109125307B - Clomidinol-polypeptide compound, pharmaceutical preparation, and preparation methods and applications thereof - Google Patents

Clomidinol-polypeptide compound, pharmaceutical preparation, and preparation methods and applications thereof Download PDF

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CN109125307B
CN109125307B CN201811020200.8A CN201811020200A CN109125307B CN 109125307 B CN109125307 B CN 109125307B CN 201811020200 A CN201811020200 A CN 201811020200A CN 109125307 B CN109125307 B CN 109125307B
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岳旭龙
李灵娟
吕凤霞
李攀登
孙江宏
李坤鹏
梁军
韩露
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Henan Muxiang Biotechnology Co ltd
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Henan Soar Veterinary Pharmaceutical Co Ltd
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis

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Abstract

The invention belongs to the technical field of treatment and/or prevention of ovulation dysfunction, and particularly discloses a clomiphene-polypeptide compound, a pharmaceutical preparation, and a preparation method and application thereof. The compound consists of clomiphene and functional polypeptide; the functional polypeptide is one or the combination of more than two of polypeptide IGp, polypeptide TGp and polypeptide PGp; the molar ratio of the clomiphene to the polypeptide IGp is 10: 1-1: 100, the molar ratio of the clomiphene to the polypeptide TGp is 1: 10-1: 200, and the molar ratio of the clomiphene to the polypeptide PGp is 1: 10-1: 100. The clomiphene-polypeptide compound can enhance the absorption of cells to drugs, increase the targeting of clomiphene to ovary and endometrial epithelial cells to a certain extent, promote follicular development and ovulation, and reduce the side effects of the clomiphene on the transition stimulation of ovaries and the thinning of endometrium.

Description

Clomidinol-polypeptide compound, pharmaceutical preparation, and preparation methods and applications thereof
Technical Field
The invention belongs to the technical field of treatment and/or prevention of ovulation dysfunction, and particularly relates to a clomiphene-polypeptide compound, a pharmaceutical preparation, and a preparation method and application thereof.
Background
Clomiphene, also known as Clomiphene Citrate (CC), is a derivative of triphenylethylene (triphenylethylene) that acts on estrogen-dependent tissues, including hypothalamus, pituitary, ovary, and endometrium, and is an antiestrogen drug with weak estrogenic effects. In the early 60 s, the medicine is applied to the clinic, and a plurality of clinical experiences are obtained, and the medicine is the most common ovulation-promoting medicine at present.
The ovulation-promoting mechanism of clomiphene is as follows: at the hypothalamic level, the estrogen competes with endogenous estrogen to bind to an estrogen receptor, and because of stronger affinity, the target organ is insensitive to the estrogen, so that the negative feedback inhibition of the estrogen to the hypothalamus is relieved, the neuroendocrine mechanism of the hypothalamus is activated, the release frequency of the hypothalamic gonadotropin releasing hormone (GnRH) is increased, the increase of the pituitary Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) levels is stimulated, and the follicular development is started to promote the growth of follicles. It has also been suggested that CC can bind to pituitary estrogen receptors and directly stimulate the release of FSH, LH. Notably, the achievement of CC ovulation-promoting effects relies on a normal HPO (hypothalamic-pituitary-ovarian) axis positive-negative feedback mechanism.
Ovulation-promoting drugs such as clomiphene and the like which are commonly used clinically can bring some side effects, such as poor cervical mucus quality, thin endometrium, delayed endometrium maturation, ovarian hyperstimulation, multiple pregnancy and the like. In the effective treatment period of the clomiphene, the ovulation rate is as high as 70-80%, but the pregnancy rate is only 30-40%. The main reason is that the local antiestrogen effect of CC results in thick cervical mucus which is not favorable for sperm to pass through, and CC is combined with the endometrial estrogen antibody to interfere the endometrial development to influence the implantation of fertilized eggs.
At present, research aiming at clomiphene mainly focuses on combined medication, ensures ovulation promoting effect and simultaneously reduces side effect, and research on clomiphene related compounds and pharmaceutical compositions is rare.
Modern research finds that: many small molecule polypeptides (e.g., gonadotropin-releasing hormone-alpha, transforming growth factor, etc.) play important roles in regulating hormone levels, ovarian stimulation, and promoting normal follicular development and ovulation. The research proves that: part of small molecular polypeptide has stronger stability, can form a nano fiber structure through self-assembly to be used as a medicine skeleton to wrap the medicine, so that the medicine has certain targeting property and slow release property, and plays the role of the small molecular polypeptide while improving the curative effect of the medicine.
Disclosure of Invention
Aiming at the problems of high ovulation, low pregnancy and the like caused by the transitional stimulation of the current clinical clomiphene to the ovary and the thinning of the endometrium, the invention aims to provide a clomiphene-polypeptide compound, a pharmaceutical preparation, a preparation method and an application thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows
A clomiphene-polypeptide complex, which complex consists of clomiphene and a functional polypeptide; the functional polypeptide is one or the combination of more than two of polypeptide IGp, polypeptide TGp and polypeptide PGp; the molar ratio of the clomiphene to the polypeptide IGp is 10: 1-1: 100, the molar ratio of the clomiphene to the polypeptide TGp is 1: 10-1: 200, and the molar ratio of the clomiphene to the polypeptide PGp is 1: 10-1: 100;
the amino acid sequence of the polypeptide IGp is shown as SEQ ID NO.1, the amino acid sequence of the polypeptide TGp is shown as SEQ ID NO.2, and the amino acid sequence of the polypeptide PGp is shown as SEQ ID NO. 3.
Further preferably, the molar ratio of the clomiphene to the polypeptide IGp is 1: 1-1: 50, the molar ratio of the clomiphene to the polypeptide TGp is 1: 20-1: 80, and the molar ratio of the clomiphene to the polypeptide PGp is 1: 50-1: 80.
More preferably, the molar ratio of the clomiphene to the polypeptide IGp is 1: 2-1: 20, the molar ratio of the clomiphene to the polypeptide TGp is 1: 25-1: 50, and the molar ratio of the clomiphene to the polypeptide PGp is 1: 65-1: 75.
A method for preparing a clomiphene-polypeptide complex: weighing clomiphene and functional polypeptide according to a molar ratio, dissolving the clomiphene and the functional polypeptide in a solvent together, stirring and mixing uniformly at 0-10 ℃, then freezing for 4-12 h at-20 to-30 ℃, then completely melting at 10-30 ℃, then ultrasonically dispersing uniformly at 0-10 ℃, stirring for 1-2 h at room temperature, and further freezing and drying at-20 to-60 ℃ to obtain a clomiphene-polypeptide compound; the solvent is water, normal saline or PBS buffer solution; the addition amount of the solvent is 2-10mL per 0.1mg of the clomiphene.
The application of the clomiphene-polypeptide compound in preparing a medicine for treating and/or preventing ovulation dysfunction.
A pharmaceutical preparation prepared from the clomiphene-polypeptide compound is prepared from the clomiphene-polypeptide compound and one or more pharmaceutically acceptable auxiliary materials.
Preferably, the pharmaceutical preparation is oral liquid, freeze-dried powder or injection; the oral liquid, the freeze-dried powder or the injection can be prepared according to the prior art, but the invention claims that the freeze-dried powder and the injection are respectively prepared according to the following processes.
The preparation process of the freeze-dried powder is as follows: taking 10-20 parts of clomiphene-polypeptide compound, 2-10 parts of water-soluble filler, 1-5 parts of stabilizer, 0.5-2 parts of osmotic pressure regulator and a proper amount of water for injection, stirring until the clomiphene-polypeptide compound, the water-soluble filler, the stabilizer, the osmotic pressure regulator and the water for injection are dissolved, adjusting the pH value to 4.0-9.0 by using the pH regulator, adding the water for injection to 100 parts, adding 0.1-0.5 part of activated carbon, stirring for 10-20 min at 0-10 ℃, removing carbon, filtering and sterilizing by using a microporous filter membrane, precooling for 4-6 h at-4-10 ℃, freeze-drying at-20-60 ℃ under reduced pressure, and drying for 1-2 h at 0-5 ℃ to prepare the freeze-dried powder.
The preparation process of the injection comprises the following steps: taking 20-50 parts of clomiphene-polypeptide compound, 10-20 parts of water-soluble filler, 2-10 parts of stabilizer, 0.5-2 parts of osmotic pressure regulator and a proper amount of water for injection, stirring until the clomiphene-polypeptide compound, the water-soluble filler, the stabilizer, the osmotic pressure regulator and the water for injection are dissolved, adjusting the pH value to 4.0-9.0 by using the pH regulator, adding the water for injection to 100 parts, adding 0.1-0.5 part of activated carbon, stirring for 10-20 min at 0-10 ℃, decarbonizing, and filtering and sterilizing by using a microporous filter membrane to obtain a filtrate, namely the injection.
The water-soluble filler of the present invention is selected from one or more of the following: mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose, galactose, etc.; the pH adjusting agent is selected from one or more of the following: non-volatile acids such as citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid, and physiologically acceptable organic or inorganic acids and bases and salts such as potassium hydroxide, sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate salt, sodium bicarbonate, potassium bicarbonate, or ammonium bicarbonate salt; the stabilizer is selected from one or more of the following: poloxamer, EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, glutamic acid, polyethylene glycol 6000, polyethylene glycol 4000, sodium dodecyl sulfate, tris (hydroxymethyl) aminomethane, etc., preferably sodium thiosulfate, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, arginine, polyethylene glycol 4000, sodium dodecyl sulfate; the osmotic pressure regulator is one or the combination of sodium chloride and potassium chloride.
The clomiphene-polypeptide complex of the invention can be administered orally, intramuscularly or subcutaneously. Although the dosage varies depending on the subject to be treated, the mode of administration, the symptoms and other factors, the clomiphene-polypeptide complex of the invention is effective over a relatively wide dosage range. In clinical treatment, when the single drug is used, the adult dose is calculated according to clomiphene/body weight per day, the single drug is taken once a day, the oral administration is taken for 2-5 mg/kg per day or the intramuscular/subcutaneous injection is taken for 1-2 mg/kg per day, and the continuous drug administration is carried out for 5 days. Specifically, for patients with ovulation dysfunction, the drug is administered at the beginning of 5d of the menstrual cycle or withdrawal bleeding for prevention, the drug is administered at the beginning of 7d of the menstrual cycle or withdrawal bleeding for treatment, and the drug is administered at 2 mg/kg. d.times.5 d by oral administration or 1 mg/kg. d.times.5 d by injection; adding the dose of non-responders (no ovulation reaction or ovulation reaction but no pregnancy) to oral administration of 3 mg/kg. d.times.5 d or injection administration of 1.5 mg/kg. d.times.5 d in the 2 nd month; adding the drug for the non-responder (no ovulation reaction or ovulation reaction but no pregnancy) in the 3 rd month to orally administer 5 mg/kg. d.times.5 d or inject 2 mg/kg. d.times.5 d; the ovulation promoting course has 3 cycles, and the treatment period is gestation and stopped.
The clomiphene-polypeptide compound can enhance the absorption of cells to drugs, increase the targeting of clomiphene to ovary and endometrial epithelial cells to a certain extent, promote follicular development and ovulation, and reduce the side effects of the clomiphene on the transition stimulation of ovaries and the thinning of endometrium.
Detailed Description
The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.
Example 1 preparation, identification and purification of functional Polypeptides IGp, TGp, PGp
The amino acid sequence of polypeptide IGp is: GPETLCGAEL VDALQFVCGD RGFYFNKPTG YGSSSRRAPQ TGIVDECCFR SCDLRRLEMY CAPLKPAKSA (shown as SEQ ID NO. 1), and the amino acid sequence of the polypeptide TGp is: VVSHFNDCPL SHDGYCLHDG VCMYIEALDK YACNCVVGYI GERCQYRDLK WWEL (shown as SEQ ID NO. 2), the amino acid sequence of the polypeptide PGp is: NSYPGCPSSY DGYCLNGGVC MHIESLDSYT CNCVIGYSGD RCEHADLLA (shown in SEQ ID NO. 3).
Amino acids protected by t-butyloxycarbonyl (Boc) were synthesized by solid-phase synthesis on an Apogee full-automatic polypeptide synthesizer (purchased from NatureGene Corp., Nature technologies, Ltd.) according to the amino acids required for the sequences shown in polypeptides IGp, TGp and PGp, respectively, according to the manufacturer's instructions. The resulting polypeptide was determined by mass spectrometric molecular weight determination, HPLC purification, and confirmed by sequencing by shanghai bio-technology corporation.
Example 2: cromiphene-polypeptide TGp compound (molar ratio 1: 30)
Weighing 0.1mg of clomiphene (MW: 598.09) dry powder according to the molar ratio of 1: 30 of clomiphene to polypeptide TGp, dissolving the clomiphene dry powder in 2ml of PBS (pH 6.0), adding 31.75mg of polypeptide TGp (MW: 6330) under stirring at 10 ℃, stirring and uniformly mixing for 20 minutes, placing the homogenate in a water bath at 20 ℃ for quick freezing at-30 ℃, freezing for 4 hours, placing the homogenate in a water bath at 20 ℃ for slow thawing, placing the homogenate in an ultrasonic bath (800W) at 4 ℃ for dispersing for 20 minutes, stirring for 1 hour at room temperature, further freezing and drying at-30 ℃ to obtain clomiphene-polypeptide compound (solid powder), and placing the clomiphene-polypeptide compound for later use at 4 ℃.
Example 3: cromiphene-polypeptide TGp complex (molar ratio 1: 15)
Weighing 0.1mg of clomiphene (MW: 598.09) dry powder according to the molar ratio of 1: 15 of clomiphene to polypeptide TGp, dissolving in 2ml of pure water, adding 15.88mg of polypeptide TGp (MW: 6330) under stirring at 4 ℃, stirring and uniformly mixing for 25 minutes, quickly freezing the homogenate at-30 ℃, placing in a water bath at 20 ℃ after freezing for 6 hours to slowly melt the homogenate, placing in ultrasound (800W) at 0 ℃ for dispersing for 20 minutes, stirring for 2 hours, further freezing and drying at-30 ℃ to obtain a clomiphene-polypeptide TGp compound (solid powder), and placing at 4 ℃ for later use.
Example 4: clomiphene-polypeptide IGp, PGp complex
Weighing 0.1mg of clomiphene (MW: 598.09) dry powder according to the molar ratio of 1: 20 of clomiphene to polypeptide IGp and the molar ratio of 1: 50 of clomiphene to polypeptide PGp, dissolving the clomiphene (MW: 598.09) dry powder in 5ml of physiological saline, adding 25.61mg of polypeptide IGp (MW: 7660) freeze-dried powder and 43.97mg of polypeptide PGp (MW: 5260) freeze-dried powder under the condition of 4 ℃, stirring and uniformly mixing for 40 minutes, placing the homogenate at-30 ℃ for quick freezing, placing the homogenate in a water bath at 30 ℃ after freezing for 12 hours to slowly melt the homogenate, placing the homogenate in an ultrasonic (200W) at 10 ℃ for 20 minutes, stirring for 1 hour, further freezing and drying at-30 ℃ to obtain a clomiphene-polypeptide IGp and PGp compound (solid powder), and placing the compound at 4 ℃ for later use.
Example 5: clomiphene-polypeptide IGp, TGp, PGp complexes
According to the molar ratio of 1: 10 of clomiphene to polypeptide IGp, the molar ratio of 1: 30 of clomiphene to polypeptide TGp and the molar ratio of 1: 70 of clomiphene to polypeptide PGp, 0.1mg of dry powder of clomiphene (MW: 598.09) is weighed, dissolved in 10ml of pure water, 12.81mg of polypeptide IGp (MW: 7660) freeze-dried powder, 31.75mg of polypeptide TGp (MW: 6330) freeze-dried powder and 61.56mg of polypeptide PGp (MW: 5260) freeze-dried powder are added under stirring at 0 ℃, stirred and mixed uniformly for 30 minutes, the homogenate is placed at-30 ℃ for quick freezing, is placed in a water bath at 30 ℃ after being frozen for 4 hours to be slowly melted, is placed in ultrasound (200W) at 10 ℃ for 20 minutes and is stirred for 1 hour, is further frozen and dried at-30 ℃ to obtain a clomiphene-polypeptide IGp, TGp and PGp compound (solid powder), and is placed at 4 ℃ for standby.
Example 6: clomiphene-polypeptide IGp, TGp, PGp complexes
According to the molar ratio of the clomiphene to the polypeptide IGp of 1: 20, the molar ratio of the clomiphene to the polypeptide TGp of 1: 15 and the molar ratio of the clomiphene to the polypeptide PGp of 1: 60, 0.1mg of dry powder of the clomiphene (MW: 598.09) is weighed, the dry powder is dissolved in 10ml of pure water, 25.61mg of polypeptide IGp (MW: 7660) freeze-dried powder, 15.88mg of polypeptide TGp (MW: 6330) freeze-dried powder and 52.77mg of polypeptide PGp (MW: 5260) freeze-dried powder are added under stirring at 0 ℃, the mixture is uniformly stirred and uniformly mixed for 35 minutes, the homogenate is placed at-30 ℃ for quick freezing, is placed in a water bath at 20 ℃ after 8 hours of freezing, is slowly melted, is placed in ultrasound (800W) at 0 ℃ for 10 minutes and is stirred for 2 hours, the clomiphene-polypeptide IGp, TGp and PGp complex (solid powder) is obtained at-30 ℃ and is placed at 0 ℃ for standby.
Example 7: preparation of lyophilized powder
Adding poloxamer 0.2g, sorbitol 0.4g, lactose 0.2g, sodium chloride 0.2g and water for injection 5ml into a proper container, stirring to dissolve, adding citric acid or sodium hydroxide solution 1mol/L to adjust pH to 6.5, adding 2.0g of the clomiphene-polypeptide TGp composite powder prepared by the method in the embodiment 3, continuously adjusting pH to 6.5, adding water for injection to 20ml, adding 20mg of activated carbon, stirring for 20 minutes at 5 ℃, decarburizing, filtering and sterilizing by using a microporous filter membrane, pre-freezing the filtrate for 4 hours at-10 ℃, freezing at 60 ℃, drying under reduced pressure for 8 hours, drying for 2 hours after the sample temperature reaches 5 ℃, obtaining a light yellow loose block, placing the light yellow loose block into a pre-filled syringe, sealing to obtain freeze-dried powder, wherein the specification is 50 mg/count, and the freeze-dried powder is stored at 4 ℃ below.
Example 8: preparation of injection
Taking a proper amount of container, adding mannitol 2.5g, polyethylene glycol 40000.5 g, sodium chloride 0.15g, and water for injection 7ml，Stirring for dissolving, adding 1mol/L citric acid or sodium hydroxide solution to adjust pH to 7.0, adding 4.0g of the compound powder of the clomiphene-polypeptide IGp, TGp and PGp prepared by the method in the embodiment 6, continuously stirring and uniformly mixing, adjusting pH to 7.0 with citric acid, adding water to 20ml, adding 100mg of activated carbon, stirring for 10 minutes at 10 ℃, decarburizing, filtering and sterilizing by adopting a microporous filter membrane, subpackaging the filtrate with 200 mu L per filter into a prefilled syringe, and storing at the temperature of below 5 ℃.
Effect of Cromiphene-polypeptide complexes on ovulation, endometrium and pregnancy rates in mice
90 female Kunming mice with the clean grade, sexual maturity, no mating, age of 8-10 weeks and weight of 30-35g are selected and randomly divided into 5 groups of 18 mice. Feeding according to normal feeding conditions, and freely taking food and drinking water. The vaginal smear was observed continuously for 3 normal estrus cycles.
The estrus cycle of the mice is 4-5 days, and the estrus cycle is divided into 4 stages: a prophase Estrus Stage (P), an Estrus Stage (E), a postestrus Stage (M), and an Estrus interval (DI). And in the period P-E, the stomach is irrigated for two days, and the medicine is administrated 1 time per day. The administration of each group is shown in Table 1.
Figure RE-258953DEST_PATH_IMAGE001
After 16-20 h of the last administration, taking 6 mice from each group, conducting cervical dislocation and sacrifice, lying on the back on an ultra-clean bench, disinfecting abdominal skin and hair with 75% alcohol by volume fraction, cutting off abdominal skin and muscle layer transversely, exposing uterus and bilateral oviducts, going upwards along the oviducts, seeing bilateral ovaries near the terminal ends, separating surrounding tissues by ophthalmic scissors, taking out the uterus, the oviducts and ovaries, removing redundant adipose tissues around the ovaries, taking out the ovaries, weighing the wet weight by an electronic balance, and calculating the ovary index, namely the relative weight (mg) of the ovaries/the body mass (g) of the mice. Under a dissecting microscope, the mouse oviduct ampulla bulge is punctured by a syringe needle, and the cumulus-oocyte complex (COC) released into a cluster is slightly squeezed. And transferring COC (cytochrome oxidase) into M2 culture solution (2-10 mL) containing 1g/L hyaluronidase by using a Pasteur capillary pipette, slightly blowing and beating to completely dissolve cumulus cells wrapping the oocytes under enzyme digestion, fully washing the oocytes, transferring the oocytes into another culture dish containing 1 mL of M16 culture solution, and counting the oocytes (namely the number of ovulations) under a microscope. The results are shown in Table 2. As can be seen from Table 2: the ovary index and the ovulation number of the compound group and the clomiphene group mice are all higher than those of the blank control group, and the compound group is higher than that of the clomiphene group, so that the clomiphene-polypeptide compound can more effectively improve the ovulation number of the mice compared with the clomiphene used alone.
Figure RE-73326DEST_PATH_IMAGE002
48-52 h after the last administration, respectively taking 6 mice in each group, removing the uterus by operation, fixing the mice by 10% formalin solution, cutting the mice into an upper section, a middle section and a lower section, and preparing a conventional HE staining sheet. The HE dyed piece is analyzed by adopting an HMIAS-2000 high-definition color medical image-text analysis system to measure the thickness of the endometrium. The rest 6 of the groups are respectively injected with 5IU HCG (human chorionic gonadotropin) in the abdominal cavity, the male and female are combined in a ratio of 1: 1, and after the combination of the cages is carried out for 14-20 h, the person who finds the vaginal suppository is determined as pregnancy. The endometrial thickness and pregnancy rate of mice in each test group are shown in Table 3. As can be seen from Table 3: the average thickness of endometrium of the compound group is higher than that of the blank control group, and the pregnancy rate is the same as that of the blank control group; the average endometrial thickness of the clomiphene group is low in the blank control group, and the pregnancy rate is lower than that of the blank control group, so that the clomiphene-polypeptide compound can effectively increase the endometrial thickness of a mouse, and the side effects of thinning the endometrium of the mouse and reducing the pregnancy rate caused by singly using the clomiphene are avoided.
Figure RE-833471DEST_PATH_IMAGE003
Sequence listing
<110> Henan Muxiang animal pharmaceutical Co., Ltd
HENAN MUXIANG BIOTECHNOLOGY Co.,Ltd.
<120> clomiphene-polypeptide compound, pharmaceutical preparation, preparation method and application thereof
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Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala Leu Gln Phe
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Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp Glu Cys Cys
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Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys Ala Pro Leu
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Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp
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Gly Gly Val Cys Met His Ile Glu Ser Leu Asp Ser Tyr Thr Cys Asn
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Cys Val Ile Gly Tyr Ser Gly Asp Arg Cys Glu His Ala Asp Leu Leu
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Ala

Claims (7)

1. A clomiphene-polypeptide complex, wherein: the compound consists of clomiphene and functional polypeptide; the functional polypeptide is IGp and PGp, and the molar ratio of the clomiphene to the polypeptide IGp to the polypeptide PGp is 1: 20: 50; or the functional polypeptide is IGp, TGp and PGp, and the molar ratio of the clomiphene to the polypeptide IGp to the polypeptide TGp to the polypeptide PGp is 1: 10: 30: 70; (ii) a
The amino acid sequence of the polypeptide IGp is shown as SEQ ID NO.1, the amino acid sequence of the polypeptide TGp is shown as SEQ ID NO.2, and the amino acid sequence of the polypeptide PGp is shown as SEQ ID NO. 3.
2. A method of preparing the clomiphene-polypeptide complex of claim 1, wherein: weighing clomiphene and functional polypeptide according to a molar ratio, dissolving the clomiphene and the functional polypeptide in a solvent together, stirring and mixing uniformly at 0-10 ℃, then freezing for 4-12 h at-20 to-30 ℃, then completely melting at 10-30 ℃, then ultrasonically dispersing uniformly at 0-10 ℃, stirring for 1-2 h at room temperature, and further freezing and drying at-20 to-60 ℃ to obtain a clomiphene-polypeptide compound;
the solvent is water, normal saline or PBS buffer solution; the addition amount of the solvent is 2-10mL per 0.1mg of the clomiphene.
3. Use of a clomiphene-polypeptide complex as claimed in claim 1 in the manufacture of a medicament for the treatment and/or prevention of ovulation dysfunction.
4. A pharmaceutical formulation prepared using the clomiphene-polypeptide complex of claim 1, wherein: the pharmaceutical preparation is prepared from the clomiphene-polypeptide compound and one or more pharmaceutically acceptable auxiliary materials.
5. The pharmaceutical formulation of claim 4, wherein: the medicinal preparation is oral liquid, lyophilized powder or injection.
6. The pharmaceutical formulation of claim 5, wherein the lyophilized powder is prepared by the process of: taking 10-20 parts of clomiphene-polypeptide compound, 2-10 parts of water-soluble filler, 1-5 parts of stabilizer, 0.5-2 parts of osmotic pressure regulator and a proper amount of water for injection, stirring until the clomiphene-polypeptide compound, the water-soluble filler, the stabilizer, the osmotic pressure regulator and the water for injection are dissolved, adjusting the pH value to 4.0-9.0 by using the pH regulator, adding the water for injection to 100 parts, adding 0.1-0.5 part of activated carbon, stirring for 10-20 min at 0-10 ℃, decarbonizing, filtering for sterilization, precooling for 4-6 h at-4 to-10 ℃, freeze-drying under reduced pressure at-20 to-60 ℃, and drying for 1-2 h at 0-5 ℃ to obtain the freeze-dried powder.
7. The pharmaceutical formulation of claim 5, wherein the injection is prepared by the process of: taking 20-50 parts of clomiphene-polypeptide compound, 10-20 parts of water-soluble filler, 2-10 parts of stabilizer, 0.5-2 parts of osmotic pressure regulator and a proper amount of water for injection, stirring until the clomiphene-polypeptide compound, the water-soluble filler, the stabilizer, the osmotic pressure regulator and the water for injection are dissolved, adjusting the pH value to 4.0-9.0 by using the pH regulator, adding the water for injection to 100 parts, adding 0.1-0.5 part of activated carbon, stirring for 10-20 min at 0-10 ℃, decarbonizing, filtering and sterilizing to obtain a filtrate, namely the injection.
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