Pharmaceutical composition for treating low ovarian function or premature ovarian failure and application thereof
By using C-type natriuretic peptide (CNP) to bind to NPR-B receptors, the increased cGMP level is promoted, and the treatment problems of premature ovarian failure or hypoovarian function is solved, and the effect of improving ovarian function and restoring organ damage is achieved.
Patent Information
- Application Number
- CN202411906993.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-04-01
AI Technical Summary
The lack of effective methods in the prior art to treat premature ovarian failure or hypoovarian function has led to women facing health problems such as infertility and menopause symptoms.
C-type natriuretic peptide (CNP) is used as a pharmaceutical composition to promote the increase in intracellular cGMP levels by binding to its specific receptor NPR-B, thereby preventing and treating hypoovarian function.
CNP can improve ovarian function, reverse organ damage caused by premature ovarian failure, restore normal functions of the ovaries and uterus, and significantly improve egg quality and blastocyst formation rate.
Smart Images
Figure CN119733035A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine, and in particular to a pharmaceutical composition for treating premature ovarian failure or ovarian dysfunction and an application thereof. Background Art
[0002] Ovarian dysfunction, also known as premature ovarian insufficiency (POI), refers to acquired hypergonadotropic hypoplasia before the age of 40. Its symptoms are abnormal menstruation (amenorrhea or oligomenorrhea) accompanied by increased gonadotropin and decreased estrogen; its diagnosis requires both abnormal menstruation and abnormal biochemical indicators: (1) oligomenorrhea / amenorrhea for more than 4 months; (2) two FSH levels of 25 IU / L (the interval between the two monitorings must be more than 4 weeks). It is estimated that the incidence of primary POI is 1-2% before the age of 40, and its incidence has been increasing in recent years. POI brings a series of health problems to women, such as infertility and menopausal symptoms, and seriously endangers women's physical and mental health. Some studies have shown that premature ovarian failure is related to genetic factors, immune factors, infectious factors and environment, but there is currently no good treatment. In addition to traditional Western medical treatments such as hormone replacement therapy and ovulation induction therapy, there are also some cutting-edge studies (such as human amniotic mesenchymal stem cell transplantation, NAD+ level enhancement, etc.) that show potential therapeutic effects on ovarian dysfunction. However, these methods are still in the experimental stage and have not yet been widely used in clinical practice.
[0003] C-type natriuretic peptide (CNP) is the third member of the natriuretic peptide family. It is a cyclic structure containing 17 amino acids connected by disulfide bonds. The first two members of the natriuretic peptide family, atrial natriuretic peptide and brain natriuretic peptide, are both powerful cardiac hormones. Studies generally believe that CNP is an endothelial activating factor, which mainly activates guanylate cyclase after binding to its specific receptor, natriuretic peptide receptor B (NPR-B), and promotes the increase of intracellular cyclic guanosine monophosphate (3'-5'-cyclic guanosine monophosphate, cGMP) levels, thereby producing its biological effects in local specific tissues in an autocrine and paracrine manner.
[0004] In recent years, the effects of CNP on reproduction have attracted everyone's attention. Many studies have reported that CNP can inhibit oocyte meiosis. Its mechanism is mainly that CNP expressed by granulosa cells in the wall of mature follicles binds to the specific receptor NPR-B in the cumulus cells around the oocytes, promotes the expression of cGMP in cumulus granulosa cells and oocytes, prevents oocytes from undergoing meiosis prematurely, and ensures the synchronization of oocyte maturation and ovulation, which plays a key role in maintaining normal female reproductive capacity. CNP-specific receptor-NPR-B is critical for the development of female reproductive organs. Female mice with NPR-B gene knockout can lead to abnormal development of their reproductive organs and infertility. Geister et al. found that after the NPR-B gene mutation, female mice had delayed puberty, reduced ovulation, increased fragments in eggs, and could not develop to the 2-cell stage. In the rat uterus, CNP and NPR-B are specifically expressed with the change of estrus cycle, and the highest expression is in the proestrus. Intraperitoneal injection of estrogen in ovariectomized mice can increase the content of uterine CNP, and it is hormone-dependent. The applicant's previous study found that CNP expression in the follicular fluid of IVF patients is related to clinical pregnancy outcomes, and CNP in vitro can significantly improve egg quality and blastocyst formation rate. Sato Y et al. found that CNP can significantly promote ovarian growth and stimulate the development of preantral follicles in infant mice. Treating prepubertal mice with CNP alone can promote the development of early antral follicles to preovulatory follicles, ovulate under the action of LH or HCG, and fertilize and produce live offspring. Therefore, CNP is expected to replace FSH and become another target for the treatment of female infertility.
[0005] Currently, clinical treatment of POI is difficult and limited, and there is still a lack of good and effective treatment methods. The applicant's previous research has confirmed that CNP can improve the reproductive function of female mice with POI model. Therefore, CNP is expected to become a potential treatment for POI. Summary of the invention
[0006] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is the lack of treatment methods for POI and how to explore new treatment methods.
[0007] To achieve the above object, the present invention provides a C-type natriuretic peptide capable of preventing and / or treating ovarian dysfunction (POI).
[0008] In a preferred embodiment of the present invention, the present invention also provides a pharmaceutical composition comprising C-type natriuretic peptide or a pharmaceutically acceptable salt thereof capable of preventing and / or treating ovarian dysfunction (POI).
[0009] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.
[0010] Preferably, the pharmaceutical composition can be formulated into an injectable fluid, an aerosol, a cream, a gel, a pill, a capsule, a syrup, or a transdermal patch.
[0011] In another preferred embodiment of the present invention, the present invention also provides a C-type natriuretic peptide capable of preventing and / or treating ovarian dysfunction (POI) and / or the use of the pharmaceutical composition in the preparation of a drug for preventing and / or treating ovarian dysfunction or premature ovarian failure.
[0012] Preferably, the prevention and / or treatment of ovarian dysfunction or premature ovarian failure is to alleviate estrous cycle disorders and repair luteal insufficiency.
[0013] In another preferred embodiment of the present invention, the present invention also provides a C-type natriuretic peptide capable of preventing and / or treating ovarian dysfunction (POI) and / or the use of the pharmaceutical composition in the preparation of a drug for preventing and / or treating ovarian, uterine, liver, and spleen damage caused by cyclophosphamide.
[0014] The present invention prepares a pharmaceutical composition capable of preventing and / or treating POI. Mouse model experiments have verified that the pharmaceutical composition can restore the general state of CTX-induced premature ovarian failure mice and reverse the damage of CTX to the morphological structure of the ovaries, uterus, liver and spleen, thereby making up for the deficiency of treatment methods for POI and having broad application value for the clinical treatment of POI or premature ovarian failure.
[0015] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 are the morphological images of the uterus and ovaries of the mouse models in the control group, CTX group, and CNP group;
[0017] Figure 2 This is the weight trend chart of the mouse models in the control group, CTX group, and CNP group;
[0018] Figure 3 It is a bar graph of the body weight of the mouse models in the control group, CTX group, and CNP group on days 0, 7, and 12;
[0019] Figure 4 is the bar graph of the liver, spleen, ovary, and uterus organ indexes of the mouse models in the control group, CTX group, and CNP group;
[0020] Figure 5 It is a vaginal mucus smear of a normal mouse estrous cycle;
[0021] Figure 6It is a bar graph of the proportion of estrous cycle disorder in the mouse model of the control group, CTX group, and CNP group;
[0022] Figure 7 These are the HE staining pictures of ovarian tissues of the mouse models in the control group, CTX group, and CNP group;
[0023] Figure 8 These are the HE staining pictures of uterine tissues of the mouse models in the control group, CTX group, and CNP group;
[0024] Fig. 9 These are the HE staining pictures of liver tissues of the mouse models in the control group, CTX group, and CNP group;
[0025] Fig.10 These are HE staining pictures of spleen tissues of mice in the control group, CTX group, and CNP group;
[0026] Fig.11 It is a bar graph of the relative expression levels of apoptotic factors and inflammatory factors mRNA in the ovarian and uterine tissues of the mouse models in the control group, CTX group and CNP group. DETAILED DESCRIPTION
[0027] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.
[0028] Example 1 Effect of CNP on Body Weight and Organ Indexes in Cyclophosphamide-Induced Premature Ovarian Failure Mouse Model
[0029] The general status of mice in each group during modeling and treatment was observed. In terms of morphology, there was no significant difference in the appearance of mice in each group. In terms of behavior, mice in the modeling group (CTX group, CNP group) were more agitated; Figure 1 As shown in the figure, the uterus of mice in the CTX and CNP groups was smaller than that in the control group, and there was no significant difference in the size of the ovaries observed with the naked eye.
[0030] The weight changes of mice in each group during the experiment were observed. Figure 2 As shown in the figure, there was no significant difference in body weight among the three groups on day 0 of the experiment. On day 12, the body weight of the control group was > that of the treatment group > that of the CTX group, but the difference was not statistically significant. Figure 3 As shown in the figure, the body weight of the CTX group and the treatment group showed a downward trend from day 0 to day 4, and the body weight of the mice in the CTX group gradually stabilized after 4 days; after the start of CNP treatment, the body weight of the mice in the treatment group increased significantly, indicating that CNP has a certain effect on the recovery of the general state of mice with CTX-induced premature ovarian failure.
[0031] Figure 4The organ index of each group of mice is shown. The ovarian and uterine indexes of the control group are the highest. The ovarian and uterine indexes of the CNP group are slightly greater than those of the CTX group, but are still significantly lower than those of the control group, indicating that CNP has a certain therapeutic effect on the ovarian and uterine damage caused by CTX, but cannot completely restore normal levels, which may be related to the short treatment time of CNP. The CTX liver index is slightly lower than that of the other two groups, but there is no significant difference between the groups. In addition, the spleen index after CNP treatment is higher than that of the CTX group, and the CTX spleen index is higher than that of the control group. This may be due to the increase in mass caused by congestion and necrosis of the spleen caused by CTX, which corresponds to the observation of dark red congested necrotic areas in the spleen of the CTX group. The spleen index of the CNP group is significantly higher than that of the control group and the CTX group, which may be due to the fact that the vasodilator effect of CNP aggravates the degree of spleen congestion and CNP has no obvious therapeutic effect on spleen necrosis caused by CTX, because in gross morphology, obvious congested necrotic areas were also found in the spleen of mice in the CNP group ( Figure 1 ).
[0032] Example 2 Effect of CNP on the estrous cycle of cyclophosphamide-induced premature ovarian failure mouse model
[0033] Figure 5 It shows that the estrous cycle of normal mice is divided into the following stages: (1) Proestrus: a large number of shed nucleated epithelial cells can be seen; (2) Estrus: many large, flat, non-nucleated keratinized squamous cells with irregular edges can be seen, without white blood cells and epithelial cells; (3) Metestrus: the number of keratinized epithelial cells decreases, and nucleated epithelial cells and white blood cells appear; (4) Diestrus: there are a large number of white blood cells and mucus. By observing the vaginal mucus smears of mice every day, it was found that the estrous cycle of the control group varied from 4 to 6 days and was regular, while the estrous cycle of some mice in the CTX group and CNP group was disordered and extended to 6 to 9 days, with proestrus and metestrus significantly prolonged, proving that intraperitoneal injection of CTX successfully caused ovarian function damage; Figure 6 The statistical results of the proportion of mice with estrous cycle disorders showed that the proportion of mice with estrous cycle disorders in the CNP group (50%) was lower than that in the CTX group (70%), indicating that CNP has a certain effect on restoring ovarian function.
[0034] Example 3 Effect of CNP on ovarian tissue morphology in cyclophosphamide-induced premature ovarian failure mouse model
[0035] Figure 7The results of HE staining of ovarian tissue are shown. It can be seen from the figure that the follicles in the control group are actively growing, with more follicles at all levels, fewer atretic cells, well-developed corpus luteum, large size, and abundant corpus luteum cells; the ovaries in the CTX group were atrophic, with a high degree of fibrosis, a decrease in the number of follicles at all levels, and an increase in the number of atretic follicles; the number of granulosa cell layers in the follicles was reduced, the granulosa cells were arranged in a disordered manner, the number of corpora lutea was small, and the development was poor. After CNP treatment, HE staining showed that the number of atretic follicles was reduced compared with the CTX group, and the corpus luteum was better developed, indicating that CNP can reverse the damaging effects of CTX on the ovarian morphology and structure to a certain extent.
[0036] Example 4 Effect of CNP on the Morphology of Other Organs and Tissues in Cyclophosphamide-Induced Premature Ovarian Failure Mouse Model
[0037] In order to further understand the effects of CNP treatment on the morphological structure of other organs in CTX model mice, HE staining sections were performed on the uterus, liver, and spleen tissues. Since the uterine tissue structure is greatly affected by the estrous cycle, the uterine tissues observed were all obtained from mice in estrus. Figure 8 It can be seen that HE staining of the cross-section of the uterus showed that the uterus of the control group was normal in size, the endometrial cells were arranged neatly, and the glands were clearly visible, while the uterus of the CTX group mice was obviously atrophied, the endometrium was thinner, the endometrial epithelial cells were arranged disorderly, the glands were reduced, and the glandular cavity was smaller; after CNP treatment, the endometrium was thicker than that of the CTX group, the cells were arranged more neatly, and the glands were clear.
[0038] observe Fig. 9 The HE-stained sections of liver tissues shown in the control group show that the hepatic cords have clear structures, the hepatic sinusoids have normal structures, the hepatocytes are evenly colored, the number of binucleated hepatocytes is large, the nuclear membrane boundaries are obvious, and the portal area structure is normal. The hepatocytes in the livers of mice in the CTX group were swollen and arranged in disorder; the hepatic cords were unclear and the structure was chaotic; the hepatic cords in the CNP group were unclear, but the structure was neater than that in the CTX group.
[0039] observe Fig.10 The HE-stained sections of spleen tissue shown in the figure show that the red and white pulp structures of the spleen of the mice in the control group were clear, the splenic nodules showed obvious light and dark areas, and the marginal zone was full of immune cells; after CTX modeling, the white pulp of the spleen of the mice was lighter in color, the germinal center was not obvious, the red pulp was dilated and congested, the boundary between the red and white pulp was not clear, the number of immune cells in the marginal zone was reduced, and no obvious structural differences were observed between the CNP group and the CTX group.
[0040] The above results show that CTX modeling has certain damage to multiple organs in mice, which is consistent with the results of previous literature studies. CNP has a certain recovery effect on the structural damage of uterus and liver caused by CTX.
[0041] Example 5 Effects of CNP on ovarian and uterine apoptosis-related indicators and inflammatory factors in cyclophosphamide-induced premature ovarian failure mouse model
[0042] Considering that CNP has a significant effect on restoring the morphological structure of ovarian and uterine tissues caused by CTX, in order to understand the underlying mechanism of CNP's therapeutic effect on CTX-induced ovarian and uterine damage, we used rt-PCR to detect the mRNA expression of ovarian and uterine-related apoptotic factors Bax, Bcl-2, caspase-3 and inflammatory factors TNF-α, IL-1β. Fig.11 As shown, compared with the Control group, the mRNA expression of Bax and Caspase-3 in the ovarian tissue of mice in the CTX group was significantly increased, and the mRNA expression of Bcl-2 was significantly decreased; the mRNA expression of the proinflammatory factor IL-1β was increased; after CNP treatment, the mRNA expression of Bax was significantly decreased, and the mRNA expression of IL-1β was decreased.
[0043] In uterine tissue, compared with the Control group, the mRNA expression of Bax in the uterine tissue of mice in the CTX group was significantly increased, and the mRNA expression of Bcl-2 was decreased; the mRNA expression of the proinflammatory factor TNF-α increased; after CNP treatment, the mRNA expression of Bax was significantly decreased, the mRNA expression of Bcl-2 was increased, and the mRNA expression of TNF-α was decreased. The above results indicate that the therapeutic effect of CNP on ovarian and uterine tissues may be produced by inhibiting apoptosis and reducing inflammatory response pathways.
[0044] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.
Claims
1. A C-type natriuretic peptide capable of preventing and / or treating ovarian dysfunction (POI).
2. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the C-type natriuretic peptide or a pharmaceutically acceptable salt thereof according to claim 1.
3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.
4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition can be formulated into injectable fluids, aerosols, creams, gels, pills, capsules, syrups, transdermal patches.
5. Use of the C-type natriuretic peptide according to claim 1 and / or the pharmaceutical composition according to any one of claims 2 to 4 in the preparation of a drug for preventing and / or treating ovarian dysfunction or premature ovarian failure.
6. The use according to claim 3, characterized in that: The prevention and / or treatment of ovarian dysfunction or premature ovarian failure is to alleviate estrous cycle disorders and repair luteal insufficiency.
7. Use of the C-type natriuretic peptide according to claim 1 and / or the pharmaceutical composition according to any one of claims 2 to 4 in the preparation of a medicament for preventing and / or treating ovarian, uterine, liver and spleen damage caused by cyclophosphamide.