Application of pharmaceutical composition in preparation of antagonistic GnRH medicine

Abstract

The invention discloses an application of a pharmaceutical composition in preparation of an antagonistic GnRH drug, the pharmaceutical composition at least comprises a compound as shown in the following formula I: # imgabs0 #, and R is CH3 or H. The compound can be JBIR-34 or JBIR-35, and the JBIR-34 or JBIR-35 has a remarkable GnRH antagonism effect and can be used for preparing a medicine for treating antagonistic GnRH. IC50 (half maximal inhibitory concentration) of GnRH is 0.8 nM and 1.1 nM respectively, and the IC50 of GnRH is obviously superior to that of a peptide GnRH antagonist cetrorelix. The pharmaceutical composition can be used for preparing drugs for treating various sex hormone-related diseases such as prostatic cancer, breast cancer, endometrial cancer, hysteromyoma, polycystic ovarian syndrome and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of a pharmaceutical composition in the preparation of a drug for treating GnRH antagonism. Background Art

[0002] Polycystic ovary syndrome (PCOS) is a common metabolic and reproductive disorder characterized by elevated androgen levels, insulin resistance (IR), and ovulatory dysfunction, all three of which are not present in all patients. These changes manifest as hyperandrogenism (such as hirsutism, acne, or alopecia, or a combination of these symptoms), oligomenorrhea or amenorrhea, and polycystic ovarian changes on ultrasound. PCOS has long been considered a reproductive disease. However, today, PCOS is considered a metabolic disease associated with long-term health risks, just like type 2 diabetes and cardiovascular disease. In addition, poor mental health outcomes and reduced quality of life are also associated with it.

[0003] Resistance of the body's cells to the effects of insulin (called insulin resistance or prediabetes) is a complication in many women with PCOS. Insulin helps sugar (glucose) enter cells so they can use it for energy. When cells resist this effect, sugar accumulates in the blood, and the pancreas produces more insulin to lower blood sugar levels. If insulin resistance is moderate or severe, diabetes may develop.

[0004] If women with PCOS who are overweight or obese become pregnant, they are at increased risk for complications during pregnancy. These complications include gestational diabetes (diabetes that develops during pregnancy), premature birth, and preeclampsia (a type of high blood pressure that develops during pregnancy).

[0005] The cause of PCOS is unknown. However, some evidence suggests that it is caused by a malfunction of an enzyme that controls male hormones. This malfunction results in high levels of male hormones.

[0006] The prevalence of PCOS is 4% to 21%. The global disease burden is growing rapidly, with the age-standardized point prevalence reported in 2019 being 1677.8 / 100,000 and the annual incidence rate being 59.8 / 100,000, an increase of 30.4% and 29.5% respectively from 1990.

[0007] PCOS is characterized by increased pulse frequency of gonadotropin-releasing hormone (GnRH) and reduced sex steroid negative feedback at the level of the hypothalamus. Neurons in the hypothalamic nucleus infundibularis release GnRH in a pulsatile manner, resulting in increased luteinizing hormone (LH) and decreased follicle-stimulating hormone (FSH) secretion. The pulse frequency of GnRH is regulated by multiple upstream endocrine and neural factors, with higher pulse frequencies favoring LH secretion and lower frequencies favoring FSH secretion. In women with PCOS, elevated LH levels lead to excess ovarian theca androgen production, while a relative lack of FSH results in follicular arrest, polycystic ovarian morphology, and infrequent ovulation. Because GnRH neurons do not have estrogen or progesterone receptors, the reduced feedback of sex steroids on GnRH release is thought to occur upstream of the hormone itself.

[0008] KNDy neurons in the arcuate nucleus of the hypothalamus co-express kisspeptin, neurokinin B (NKB) and dynorphin (DYN), which are involved in the reproductive endocrine regulation of animals and play an important role in the pulsatile secretion of gonadotropin-releasing hormone (GnRH) / luteinizing hormone (LH).

[0009] The neuronal activity of GnRH is also regulated by other substances, including gamma-aminobutyric acid (GABA) and anti-Müllerian hormone (AMH), both of which directly stimulate GnRH neurons. GABA exerts an excitatory effect on GnRH neurons through GABAA receptors, and GABA levels in the cerebrospinal fluid of PCOS patients may be increased. AMH is secreted by ovarian granulosa cells, and elevated AMH levels in PCOS women can disrupt follicle formation and ovulation. AMH may also have a neuroendocrine effect: 50% of GnRH neurons in mice and humans express AMH type 2 receptors (AMHR2), and studies have shown that AMH is associated with GnRH neuronal migration, GnRH pulsatile release, and LH secretion.

[0010] High levels of androgens increase the risk of metabolic syndrome (with high blood pressure, high cholesterol levels, and resistance to the effects of insulin) and obesity. If androgen levels remain high, the risk of diabetes, cardiovascular disease (including atherosclerosis and coronary heart disease), and high blood pressure increases. Also, some of the androgen may be converted to estrogen, raising estrogen levels. If not enough progesterone is produced to balance the elevated estrogen levels, and this condition continues for a long time, the lining of the uterus (endometrium) may become extremely thick (called endometrial hyperplasia) or develop cancer of the uterine lining (endometrial cancer). PCOS may also increase the risk of nonalcoholic fatty liver disease (abnormal accumulation of fat within liver cells, unrelated to alcohol consumption).

[0011] Currently, there are many peptide GnRH antagonists on the market, but there are problems to be solved such as oral absorption, drug stability, sustained effect and metabolic stability. Summary of the invention

[0012] The purpose of the present invention is to overcome the defects of existing polypeptide GnRH antagonists having a large number of limitations and to provide a pharmaceutical composition with good activity as a GnRH receptor antagonist for effectively treating diseases related to sex hormones.

[0013] In order to achieve the above object, the present invention provides a use of a pharmaceutical composition in the preparation of a drug for treating GnRH antagonism, wherein the pharmaceutical composition comprises at least a compound represented by the following formula I:

[0014]

[0015] Wherein, R is CH3 or H.

[0016] Optionally, the compound is JBIR-34, and its chemical structure is shown in the following formula II:

[0017]

[0018] Optionally, the compound is JBIR-35, and its chemical structure is shown in the following formula III:

[0019]

[0020] Optionally, in the GnRH antagonist drug, the dosage of the compound is 1 mg to 50 mg.

[0021] Optionally, in the GnRH antagonist drug, the dosage of JBIR-34 is 10 mg to 30 mg.

[0022] Optionally, in the GnRH antagonist drug, the dosage of JBIR-35 is 25 mg to 50 mg.

[0023] Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0024] Optionally, the pharmaceutically acceptable excipients include at least any one of a filler, a disintegrant, a glidant, a lubricant, and a coating agent, or a combination of any two or more thereof.

[0025] Optionally, the GnRH antagonist drug is in the form of any one of tablets, capsules, and granules.

[0026] Optionally, the application is for preparing drugs for treating prostate cancer, breast cancer, endometrial cancer, uterine fibroids, and polycystic ovary syndrome.

[0027] Compared with the prior art, the beneficial effects of the present invention include at least:

[0028] The pharmaceutical composition of the present invention comprises at least a compound represented by the following formula I:

[0029]

[0030] Where R is CH 3 Or H. The compound may be JBIR-34 or JBIR-35, which has a significant GnRH antagonistic effect and can be used to prepare a drug for treating GnRH antagonism. 50 The results are as follows: 0.8nM and 1.1nM, respectively, which are significantly due to the peptide GnRH antagonist cetrorelix. The inhibition rates of testosterone in the serum of rats given GnRH were 75.2% and 52.7%, respectively. For PCOS model rats, the body weight, ovarian index and uterine index of the model animals can be reduced to normal levels, the levels of LH and LH / FSH in serum can be effectively reduced, the expression of related genes in the hypothalamus, hippocampus and ovaries of the model animals can be effectively improved, and the embryonic development level of the model animals can be finally improved. The pharmaceutical composition formed by the combination of the compound shown in the formula I and the pharmaceutically acceptable excipients can be used to prepare drugs for various diseases related to sex hormones such as prostate cancer, breast cancer, endometrial cancer, uterine fibroids, polycystic ovary syndrome, etc. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below in conjunction with embodiments.

[0032] The experimental materials not specifically described in the present invention are all commercially available, and the experimental methods not specifically described in the present invention are all conventional experimental methods in the art.

[0033] Peptide GnRH receptor antagonists include cetrorelix and ganirelix, which mainly block the binding of GnRH to its receptor by rapid competitive binding to GnRH receptors, blocking the formation of dimer complexes and signal transmission, thereby inhibiting the release of LH and FSH. However, there are many problems to be solved for peptide compounds, including oral absorbability, dosage form, dosage volume, drug stability, sustained action and metabolic stability. The main reason why small molecule GnRH receptor antagonists are superior to existing peptide therapies is that small molecule GnRH receptor antagonists can be directly administered orally, which is convenient and fast. Studies have shown that small molecule antagonists have significant therapeutic effects on hormone-dependent diseases such as endometriosis, precocious puberty, and prostate cancer.

[0034] In view of the limitations of peptide GnRH receptor antagonists, some non-peptide GnRH receptor antagonists have been proposed and entered the development, clinical trial and marketing stages. For example, Elagolix developed by AbbVie has been approved for the treatment of endometriosis, and the indication of uterine fibroids has been submitted for marketing; Relugolix developed by Takada has been approved for the treatment of uterine fibroids, and has entered the Phase III clinical stage for the indications of endometriosis and prostate cancer; Linzagolix jointly developed by ObsEva and Kissei is in the Phase III clinical trial stage for the two indications of endometriosis and uterine fibroids.

[0035] Although a lot of research has been conducted in this field, there is still a need to continue to develop more effective GnRH receptor antagonists to treat various sex hormone-related diseases.

[0036] In 2010, the Journal of Natural Products published the research results of Keiichiro Motohashi et al., Tetrapeptides Possessing a Unique Skeleton, JBIR-34and JBIR-35, Isolated from a Sponge-Derived Actinomycete, Streptomyces sp.Sp080513GE-23 (Chinese translation: Tetrapeptides JBIR-34 and JBIR-35 with unique skeletons isolated from sponge actinomycete Sp080513GE-23), J.Nat.Prod.2010,73,226–228.

[0037] The document discloses the chemical structures of tetrapeptides JBIR-34 and JBIR-35, as shown in the following formula I:

[0038]

[0039] Where R = CH 3 When R=H, it is JBIR-34, and when R=H, it is JBIR-35.

[0040] The document also discloses a method for separating the above two tetrapeptide compounds, as follows:

[0041] The fermentation broth (1 L) was centrifuged, and the supernatant was layered with ethyl acetate (1 L × 3) and n-butanol (1 L × 2) in sequence. The n-butanol layer was evaporated to dryness. The dried residue (563.4 mg) was fractionated by reverse phase medium pressure preparative liquid chromatography (MPLC) using a methanol-water gradient system (0% to 100% methanol), and the fractions including the main metabolites were collected by LC-MS monitoring. The eluate was treated with preparative reverse phase HPLC (L-column 2 ODS using 65% MeOH methanol aqueous solution containing 0.1% formic acid for color development) to obtain JBIR-34 (1, tR = 19.6 minutes, 0.39 mg) and JBIR-35 (2, tR = 10.5 minutes, 0.44 mg). (ODS is dodecylsilane bonded silica gel).

[0042] The document also discloses the infrared, UV, H The above chemical structure was confirmed by NMR (hydrogen nuclear magnetic resonance) and mass spectrometry data.

[0043] Finally, the document discloses the scavenging activity of the above two tetrapeptide compounds on DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radicals. The experiments show that the IC values ​​of JBIR-34 and JBIR-35 are 50 The values ​​were 1.0 mM and 2.5 mM, respectively, which were weaker than R-tocopherol (IC50) 50 μM. ) In addition, JBIR-34 and JBIR-35 showed no cytotoxicity against several cancer cell lines and no antibacterial activity against Micrococcus luteus and Escherichia coli.

[0044] JBIR-34, molecular formula: C 21 H 25 C1N 4 O 7 , Chemical name: (2S)-2-[[(2R)-2-[[(4R)-2-(6-chloro-4-hydroxy-1,5-dimethylindol-3-yl)-4-methyl-5H-1,3- [4-oxazole-4-carbonyl]amino]propionyl]amino]-3-hydroxypropionic acid relative molecular mass: 480.9 g / mol, PubChem CID: 46182908.

[0045] JBIR-35, molecular formula: C 20 H 23 C1N 4 O 7 , Chemical name: (2S)-2-[[(2R)-2-[[(4R)-2-(6-chloro-4-hydroxy-1-methylindol-3-yl)-4-methyl-5H-1,3- [4-oxazole-4-carbonyl]amino]propionyl]amino]-3-hydroxypropionic acid, relative molecular mass: 466.9 g / mol, PubChem CID: 46182909.

[0046] The present invention provides an application of a pharmaceutical composition in preparing a drug for treating GnRH antagonism. In addition to JBIR-34 or JBIR-35, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, wherein the excipient may be any one of a filler, a disintegrant, a glidant, a lubricant, and a coating agent, or a combination of any two or more thereof. The filler is any one or more of lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, and starch; the disintegrant is any one or more of sodium carboxymethyl starch, cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone, and an effervescent disintegrant; the glidant is micropowder silica gel; and the lubricant is any one or more of magnesium stearate, stearic acid, calcium stearate, zinc stearate, and sodium stearyl fumarate.

[0047] Example 1 In vitro GnRH antagonism of JBIR-34 and JBIR-35

[0048] The competitive GnRH antagonistic effect of JBIR-34 and JBIR-35 of the present invention was evaluated by measuring the radioactivity of samples after different compounds competitively bound to human GnRH receptors with 125I-D-Trp6-LHRH.

[0049] 1.1. Preparation of CHO (Chinese Hamster Ovary) cells expressing human GnRH receptor

[0050] First, CHO cells were cultured (culture medium: α-MEM, 10% FCS, containing antibiotics and antifungal agents), and an expression vector containing the human GnRH receptor gene and the transfection reagent FuGENE6 were added. The transfection was carried out by reacting for 24 hours to obtain CHO cells that stably expressed the human GnRH receptor. The expression of the target receptor was confirmed by PCR.

[0051] 1.2. Preparation of CHO cell membrane fractions containing human GnRH receptors

[0052] In the CHO cells expressing human GnRH receptor prepared as described in step 1 (3 × 10 8100 ml of homogenization buffer (10 nM NaHCO3, 5 mM EDTA (ethylenediaminetetraacetic acid), pH 7.5) was added to the cell pellet, homogenized with a Polytron homogenizer and centrifuged at 400 × G for 15 minutes. The supernatant was centrifuged at 100,000 × G for 1 hour in an ultracentrifuge tube to obtain the membrane precipitate. The precipitate was suspended in 60 ml of storage buffer (25 mM Tris-Hcl, 1 mM EDTA, 10 ug / ml protease inhibitor), 1 ug / ml pepstatin A, 20 ug / ml leupeptin, 0.03% sodium azide, pH 7.5), aliquoted, and stored at -80 ° C. Thaw before each use.

[0053] 1.3. Yes 125 ID-Trp 6 -Determination of inhibition rate of LHRH binding

[0054] The CHO cell membrane site containing human GnRH receptor prepared in step 2 was diluted to 20ug / ml with a buffer solution (HBSS (Hanks balanced salt solution), 20mM HEPES, 0.1% BSA (bovine serum albumin), 100ug / ml bacitracin, pH 7.4) and dispensed into test tubes, 148ul per tube. Then, 2ul of the test compound dissolved in DMSO and 0.1nM of 125 ID-Trp 6 - LHRH (luteinizing hormone-releasing hormone labeled with iodine-125 isotope at position 6 of D-tryptophan) (50ul), start the reaction.

[0055] In addition, to determine the maximum binding amount, a reaction solution with DMSO (2ul) added instead of the test compound was prepared, and to determine the nonspecific binding amount, a reaction solution with 100umL HRH (2ul) (hypothalamic-releasing hormone) added instead of the test compound was prepared. These reaction solutions were incubated at 4°C for 3 hours. After incubation, the reaction solution was filtered through a Whatman glass funnel (GF / B) treated with 0.5% polyethyleneimine to stop the reaction. The radioactivity remaining on the filter paper was measured with a counter. Then PMB was calculated using the following formula:

[0056] PMB%=(SB-NSB) / (TB-NSB)×100%

[0057] Where, TB is the maximum binding radioactivity;

[0058] SB is the radioactivity when the test compound is added;

[0059] NSB is nonspecifically bound radioactivity.

[0060] Calculate the binding inhibition rate (%, PMB) of the test compound at different concentrations, plot the PMB% of the test compound at different concentrations, and find the test compound concentration when the PMB% is 50% (IC 50 The results are shown in Table 1 below:

[0061] Table 1 IC of compounds JBIR-34 and JBIR-35 for GnRH antagonism 50

[0062] Compound <![CDATA[IC 50 (nM)]]> JBIR-34 0.8 JBIR-35 1.1 Cetrorelix (positive control) 1.2

[0063] Experimental results: Both JBIR-34 and JBIR-35 have excellent GnRH antagonistic activity, which is better than the approved GnRH antagonist cetrorelix, and the antagonistic activity of JBIR-34 is particularly good. This shows that the compounds JBIR-34 and JBIR-35 of the present invention have GnRH antagonistic effects, and thus have the effect of reducing sex hormone levels, and are effective in treating sex hormone-dependent diseases such as prostate cancer, breast cancer, endometriosis, uterine fibroids and polycystic ovary syndrome.

[0064] Example 2 Test of the Antagonistic Effects of JBIR-34 and JBIR-35 on the GnRH-Induced Increase in Blood Testosterone

[0065] GnRH antagonists can directly bind to GnRH receptors and will not lead to an increase in testosterone.

[0066] The antagonistic effects of JBIR-34 and JBIR-35 on gonadotropin-releasing hormone in vivo were evaluated by their inhibitory effects on the increase in blood testosterone induced by GnRH administration in rats.

[0067] 9-week-old Wistar male rats were used. GnRH (30 ng / rat) was administered via the muscles of the buttocks of the rats. The test compound was dissolved or suspended in a 0.1% carboxymethylcellulose aqueous solution and administered orally at 10 mg / kg 3 hours before the administration of GnRH. Blood was collected 1 hour after the administration of GnRH, and the testosterone concentration in the serum was determined by a specific radioimmunoassay (RIA kit).

[0068] Assuming that the testosterone concentration in the serum of rats not given GnRH is Tn, the testosterone concentration in the serum of rats not given the test compound but given the solvent is Tc, and the testosterone concentration in the serum of rats given the test compound is Ts, the inhibitory activity (%) (IA) of the test compound is calculated according to IA% = (Tc-Ts) / (Tc-Tn) × 100% (IA = 100% when Ts decreases to Tn). The experimental results are shown in Table 2 below:

[0069] Table 2 Inhibitory activity of compounds JBIR-34 and JBIR-35 on blood testosterone levels in rats given intramuscular GnRH

[0070] Compound IA% JBIR-34 75.2％ JBIR-35 52.7％

[0071] Experimental results: JBIR-34 and JBIR-35 have an inhibitory effect on the increase of testosterone in the blood of Wistar rats given GnRH, and the antagonistic activity is greater than 50%. In particular, JBIR-34 has a better effect. It proves that JBIR-34 and JBIR-35 have excellent GnRH antagonistic activity after oral administration.

[0072] The tests in Examples 1 and 2 above indicate that JBIR-34 and JBIR-34 have a strong GnRH antagonistic effect and are useful for the treatment of prostate cancer, PCOS, breast cancer, endometriosis and uterine fibroids.

[0073] Example 3: Pharmacological Study of JBIR-34 and JBIR-35

[0074] 3.1 Modeling (dihydrotestosterone) and drug administration

[0075] Adult C57BL / 6J female mice were paired with male mice at 16:00 in the afternoon. Mice with mating plugs were identified the next morning and housed individually. Pregnant mice were injected subcutaneously (sc) with 100 μL sesame oil (vehicle control) or sesame oil containing 200 μg dihydrotestosterone (DHT) on days 16, 17, and 18 of pregnancy.

[0076] Female pups born from pregnant mice injected with dihydrotestosterone (DHT) were selected, and vaginal secretions were collected from the mice at 8:00 a.m. every day when the pups were 8-10 weeks old. Vaginal smears were taken from the mice for 2-3 cycles to monitor the estrous cycle. 80 mice with persistent keratinization of the vaginal epithelium (irregular estrous cycle, i.e., successful modeling) were selected.

[0077] Subsequently, the successfully modeled mice were randomly divided into 8 groups, namely, model control group, Diane-35 (ethinyl estradiol cyproterone acetate tablets) group (0.3392 mg / kg, 10 times the clinical dosage, 0.5% CMC was used to prepare 0.03392 mg / mL Diane-35 suspension before use, 1 mL was administered to each 100 g animal), JBIR-34 and JBIR-35 high-, medium-, and low-dose groups (0.5% CMC was used to prepare JBIR-34 suspensions of 0.18 mg / mL, 0.36 mg / mL, and 0.54 mg / mL, respectively, before use, 0.45 mg / mL, 0.72 mg / mL,

[0078] 0.9 mg / mL JBIR-35 suspension, 1 mL per 100 g animal), 10 mice per group. Except for the blank control group and the model control group which were given the same amount of solvent, the experimental mice in each treatment group were given the corresponding drugs by gavage for 3 consecutive weeks.

[0079] Animal welfare: The animals were fed and watered at 8 a.m. every day. The animals' activity, fur color, and bedding moisture were observed at 6 p.m. The bedding was changed every 3 days. The animals were fasted but not watered at 8 p.m. the day before the experiment.

[0080] 3.2 Observation indicators

[0081] 3.2.1 Observation of embryonic development

[0082] Oocyte collection: After drug administration, the mice were dissected and the oviducts were taken to provide oocytes.

[0083] Sperm collection and capacitation: Select male mice in good condition aged 14-24 weeks, kill them by cervical dislocation, remove the epididymis, squeeze out the sperm mass with fine forceps, pick out and discard the waste tissue blocks, and then put the sperm capacitation dish into the incubator for capacitation for about 1 hour.

[0084] In vitro fertilization and embryo observation: The steps are as follows:

[0085] Step 1: The female mouse was killed by cervical dislocation, the oviduct was cut, the egg mass was picked out in mineral oil, and introduced into the insemination drop;

[0086] Step 2: 1 hour after sperm capacitation, add an appropriate amount of sperm from the edge of the capacitation drop to the fertilization drop for in vitro fertilization. In vitro fertilization process: The large and round ones are egg cells, the swimming ones are sperm with fertilization ability, and the small granular ones are granulosa cells;

[0087] Step 3: After 6-7 hours, the embryos are picked out from the fertilization drop, cleaned, and cultured in a single potassium optimized medium (KSOM). The pronuclei can be observed on the same day or the development of the 2-cell embryos can be observed the next day. On the second day, the 2-cell embryos in good condition are selected for statistics, and the fertilization rate is calculated (fertilization rate = number of 2-cell embryos / total number of eggs involved in fertilization). The day of fertilization is D0.5 day, and the embryo status is observed every day until the 6th day.

[0088] 3.2.2 Body weight and organ index

[0089] When dissecting mice, surgical instruments were used to carefully separate the abdominal aorta, and blood was collected using disposable blood collection needles and blood collection tubes. After centrifugation using a high-speed refrigerated centrifuge, serum was quickly collected and the levels of LH and FSH in the serum were determined. After blood was collected from the abdominal aorta, the mice were killed by cervical dislocation, the ovaries and uterus were removed, and the fat attached to the tissues was carefully peeled off. The wet weight of each organ was recorded after weighing, and the relevant organ index was calculated.

[0090] The ovarian organ index was calculated according to the mouse body weight, that is, ovarian mass (mg) / body weight (g) × 10 -3 , and the uterine index is calculated in the same way.

[0091] 3.2.3RT-PCR

[0092] Mouse hippocampus, ovary, and hypothalamus tissue samples were collected respectively, placed in 2 ml centrifuge tubes, frozen, and set aside for subsequent PCR detection of related genes.

[0093] 3.3 Serum LH and FSH kit determination method:

[0094] Method for obtaining serum: When collecting blood from the abdominal aorta of experimental animals, caution should be exercised during the operation to prevent the blood collection needle from piercing the abdominal aorta, causing blood to splash and waste samples. After the blood is collected, place it in a 4°C cool cabinet to stand, wait for the serum on the upper part of the blood collection tube to clarify, and then put it into a high-speed refrigerated centrifuge for centrifugation. After centrifugation, the operator wears disposable gloves and quickly collects the serum. The operation process should be cautious. The collected serum samples are strictly measured according to the steps of the enzyme-linked immunosorbent assay (ELISA) kit: serum LH and FSH content.

[0095] 3.4 RT-PCR detection

[0096] Total RNA was extracted using TRIsoln reagent, and 2 μg of RNA was reverse transcribed using the Verso cDNA synthesis kit with Oligo-dT primers. Real-time quantitative polymerase chain reaction (qPCR) was performed using the Quantstudio Real-Time PCR System (Life Technologies, USA). Primers were purchased from Wuhan Jinkairui Bioengineering Co., Ltd. and are shown in Table 3. All samples were repeated three times, accompanied by a no-template control. PCR was performed using SYBR Select PCR Master Mix. Thermal cycling conditions included: 1 cycle of initial denaturation at 95°C for 15 minutes; followed by 45 cycles, each cycle including 15 seconds at 94°C, 30 seconds at 60°C, and 30 seconds at 72°C. The fold change of mRNA for each sample group was calculated using the method.

[0097] Table 3 Primer sequences

[0098]

[0099]

[0100] 3.5 Experimental Results

[0101] 3.5.1 Effects of DHT on body weight and organ indexes in PCOS model mice

[0102] The effects of DHT on the ovarian and uterine indexes of PCOS model mice are shown in Table 4.

[0103] Table 4 Effects of JBIR-34 and JBIR-35 on body weight, ovarian and uterine indexes of mice in DHT-induced PCOS model

[0104]

[0105]

[0106] From the results in Table 4, we can see that compared with the blank control group, the body weight, ovarian index and uterine index of the model control group mice were significantly increased (P<0.01), which is consistent with the ovarian weight gain of PCOS patients. Compared with the model control group, Diane-35 and large, medium and small doses of JBIR-34 and JBIR-35 can significantly reduce the body weight, ovarian and uterine index of PCOS mice (P<0.01), and the reduced levels are similar to those of the blank control group, proving that JBIR-34 and JBIR-35 can reduce the body weight, ovarian and uterine index of PCOS mice to normal levels.

[0107] 3.5.2 Effects of DHT on serum LH and LH / FSH ratio in PCOS mice

[0108] Table 5 Effects of JBIR-34 and JBIR-35 on serum LH and LH / FSH ratio in DHT-induced PCOS model mice

[0109]

[0110]

[0111] The results in Table 5 show that compared with the blank control group, the levels of LH and LH / FSH in the serum of the model control group mice were significantly increased (P<0.01). Compared with the model control group, Diane-35 and large, medium and small doses of JBIR-34 and JBIR-35 can significantly reduce the serum LH and LH / FSH levels of PCOS mice (P<0.01).

[0112] 3.5.3 Observation of embryonic development of each group of experimental mice

[0113] Table 6 Effects of JBIR-34 and JBIR-35 on embryonic development in DHT-induced PCOS model mice

[0114]

[0115]

[0116] As shown in the data in Table 6 above, DTH caused significant changes in the embryonic development-related indicators of PCOS model mice. However, after administration of positive drugs (Diane-35), JBIR-34 and JBIR-35, the embryonic development indicators of mice in each medication group tended to be normal, and JBIR-34 and JBIR-35 were both better than Diane-35.

[0117] 3.4.3 Effects of DHT on the expression of related genes in the hypothalamus, hippocampus and ovary of PCOS

[0118] Table 7 Effects of JBIR-34 and JBIR-35 on the expression of PCOS-related genes in the hypothalamus of DHT-induced PCOS

[0119] Gene n AHr ESR1 G JZ Kiss1 Kiss1r Pdyn PcqI SIC6D TAC2 Blank control 10 1 1 1 1 1 1 1 1 1 Model comparison 10 0.845 0.451 0.139 0.419 0.371 2.238 0.635 9.538 6.899 TAIE-35 group 10 0.929 0.915 0.857 0.846 0.699 1.572 0.896 5.140 4.427 JBIR-34 high dose group 10 0.956 0.937 0.921 0.901 0.809 1.475 0.926 3.405 2.322 JBIR-34 medium dose group 10 0.948 0.909 0.895 0.858 0.802 1.650 0.910 4.346 2.819 JBIR-34 low dose group 10 0.934 0.896 0.866 0.784 0.712 1.837 0.886 4.525 2.916 JBIR-35 high dose group 10 0.901 0.926 0.863 0.880 0.719 1.445 0.914 4.159 2.607 JBIR-35 medium dose group 10 0.890 0.912 0.831 0.803 0.642 1.529 0.890 5.084 3.471 JBIR-35 low dose group 10 0.873 0.860 0.706 0.752 0.476 1.871 0.852 5.212 4.224

[0120] Table 8 Effects of JBIR-34 and JBIR-35 on the expression of PCOS-related genes in the hippocampus of DHT-induced PCOS

[0121]

[0122]

[0123] Table 9 Effects of JBIR-34 and JBIR-35 on the expression of PCOS-related genes in DHT-induced PCOS ovaries

[0124] Gene n AHr ESR1 G JZ Kiss1 Kiss1r Pdyn PcqI SIC6D TAC2 Blank control 10 1 1 1 1 1 1 1 1 1 Model comparison 10 14.874 8.180 0.273 0.393 0.163 0.430 0.332 0.491 0.769 TAIE-35 group 10 8.530 4.850 0.886 0.791 0.662 0.776 0.832 0.725 0.853 JBIR-34 high dose group 10 6.130 1.543 0.956 0.972 0.879 0.974 0.985 0.975 0.980 JBIR-34 medium dose group 10 7.467 2.695 0.929 0.878 0.831 0.888 0.875 0.955 0.902 JBIR-34 low dose group 10 8.292 3.825 0.948 0.770 0.807 0.877 0.835 0.857 0.889 JBIR-35 high dose group 10 7.707 2.152 0.942 0.863 0.827 0.935 0.957 0.908 0.949 JBIR-35 medium dose group 10 8.548 3.134 0.878 0.733 0.846 0.875 0.880 0.862 0.908 JBIR-35 low dose group 10 9.110 4.113 0.858 0.665 0.766 0.794 0.860 0.754 0.834

[0125] As shown in Tables 7-9, compared with the blank control group, the expression of PCOS-related genes in the hypothalamus, hippocampus and ovary of DHT-induced PCOS model mice showed greater fluctuations, specifically manifested in greater fluctuations in the mRNA levels of related genes. After oral administration of JBIR-34 or JBIR-35 or positive drugs (Diane-35), compared with the model control group, the expression levels of related genes in each medication group tended to be normal, and JBIR-34>JBIR35>Diane-35.

[0126] Example 4 Preparation of JBIR-34 Oral Tablets

[0127] Table 10 JBIR-34 oral tablet formulation

[0128]

[0129]

[0130] As shown in Table 10, the preparation process is as follows:

[0131] Step 1) taking JBIR-34, mannitol, microcrystalline cellulose, and cross-linked sodium carboxymethyl cellulose, sieving, crushing, passing through a 40-mesh sieve, removing lumps, and setting aside;

[0132] Step 2) Take the prescribed amount of JBIR-34, mix it with microcrystalline cellulose and mannitol in equal and increasing amounts, and mix well;

[0133] Step 3) Take the material obtained in step 2), add purified water to make a soft material, granulate with a 24-mesh sieve, vacuum dry, sieve with a 40-mesh sieve to obtain drug-loaded granules for later use;

[0134] Step 4) taking the drug-loaded particles obtained in step 3), sequentially mixing with cross-linked sodium carboxymethyl cellulose and micro-powdered silica gel, and mixing evenly;

[0135] Step 5) Take the material obtained in step 4), add the prescribed amount of magnesium stearate, and mix well;

[0136] Step 6) The material obtained in step 5) is further compressed into tablets.

[0137] Example 5 Preparation of JBIR-35 Oral Tablets

[0138] Table 11 JBIR-35 oral tablet formulation

[0139]

[0140] As shown in Table 11, the preparation process is as follows:

[0141] Step 1) taking JBIR-35, mannitol, microcrystalline cellulose, and cross-linked sodium carboxymethyl cellulose, sieving, crushing, passing through a 40-mesh sieve, removing lumps, and setting aside;

[0142] Step 2) Take the prescribed amount of JBIR-35, mix it with microcrystalline cellulose and mannitol in equal and increasing amounts, and mix well;

[0143] Step 3) Take the material obtained in step 2), add purified water to make a soft material, granulate with a 24-mesh sieve, vacuum dry, sieve with a 40-mesh sieve to obtain drug-loaded granules for later use;

[0144] Step 4) taking the drug-loaded particles obtained in step 3), sequentially mixing with cross-linked sodium carboxymethyl cellulose and micro-powdered silica gel, and mixing evenly;

[0145] Step 5) Take the material obtained in step 4), add the prescribed amount of magnesium stearate, and mix well;

[0146] Step 6) The material obtained in step 5) is further compressed into tablets.

[0147] In summary, JBIR-34 and JBIR-35 of the present invention have significant GnRH antagonistic effects and can be used to prepare drugs for antagonizing GnRH. 50 The results show that the inhibition rates of testosterone in serum of rats given GnRH are 75.2% and 52.7%, respectively. For PCOS model rats, the body weight, ovarian index and uterine index of the model animals can be reduced to normal levels, the levels of LH and LH / FSH in serum can be effectively reduced, the expression of related genes in the hypothalamus, hippocampus and ovary of the model animals can be effectively improved, and the embryonic development level of the model animals can be finally improved. The pharmaceutical composition of the present invention can be used for the preparation of treatments for diseases related to sex hormones.

[0148] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. Use of a pharmaceutical composition in the preparation of a drug for treating GnRH antagonism, characterized in that: The pharmaceutical composition comprises at least a compound represented by the following formula I: Wherein, R is CH3 or H.

2. The use according to claim 1, characterized in that The compound is JBIR-34, and its chemical structure is shown in the following formula II:

3. The use according to claim 1, characterized in that The compound is JBIR-35, and its chemical structure is shown in the following formula III:

4. The use according to claim 1, characterized in that In the GnRH antagonist drug, the dosage of the compound is 1 mg to 50 mg.

5. The use according to claim 2, characterized in that In the GnRH antagonist drug, the dosage of JBIR-34 is 10 mg to 30 mg.

6. The use according to claim 3, characterized in that In the GnRH antagonist drug, the dosage of JBIR-35 is 25 mg to 50 mg.

7. The use according to claim 1, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

8. The use according to claim 7, characterized in that The pharmaceutically acceptable excipients include at least any one of a filler, a disintegrant, a glidant, a lubricant, and a coating agent, or a combination of any two or more thereof.

9. The use according to claim 1, characterized in that The GnRH antagonist drug is any one of tablets, capsules and granules.

10. The use according to claim 1, characterized in that The application is application in preparing medicines for treating prostate cancer, breast cancer, endometrial cancer, uterine fibroids and polycystic ovary syndrome.

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