Use of sodium carbenoxolide in the preparation of a medicament for the treatment of nlrp2 gene deficiency infertility

Abstract

The application belongs to the technical field of infertility treatment, and discloses application of sodium carbenoxolide in preparation of a medicine for treating NLRP2 infertility caused by gene defects. The scheme can significantly improve the blastocyst formation rate of superovulated mice, and under natural conditions, according to the different stages of estrus cycle, the number of live births shows an upward trend after accurate drug administration. At the same time, the maternal liver function and offspring growth and development safety evaluation are good. Further verification in a cynomolgus monkey model with low embryonic development rate shows that the scheme can significantly improve the blastocyst development rate. The application provides a cross-species treatment strategy for infertility caused by maternal effect gene mutations represented by Dazl, and provides a treatment strategy for infertility and insufficient oocyte development caused by maternal effect gene deletion or insufficient function. NLRP2 NLRP2 The application provides a cross-species treatment strategy for infertility caused by maternal effect gene mutations represented by Dazl, and provides a treatment strategy for infertility and insufficient oocyte development caused by maternal effect gene deletion or insufficient function.​

Description

Technical Field

[0001] This invention belongs to the field of infertility treatment technology, and more specifically, relates to the preparation of sodium glycinate for treatment. NLRP2 Application in drugs for infertility caused by genetic defects. Background Technology

[0002] Infertility refers to the inability to establish pregnancy after 12 months of normal, unprotected sexual intercourse. Early embryonic arrest is one of the main causes of human infertility. Genetic factors play a significant role in infertility, with the NLRP2 gene mutation having a detection rate of approximately 1.2% in genetically related infertility in my country.

[0003] NLRP2 (NACHT, LRR, and PYD domains-containing protein 2) is a member of the NOD-like receptor (NLR) family and participates in innate immune regulation as a component protein of the inflammasome. Recent studies have found that NLRP2 is highly expressed in mammalian oocytes and early embryos, potentially involved in key biological processes such as embryonic genome activation and epigenetic reprogramming. However, the specific function of NLRP2 in ovarian follicle development and oocyte maturation remains unclear, and research on the etiology of infertility caused by NLRP2 mutations is still limited.

[0004] Currently, clinical treatment for infertility related to genetic factors mainly relies on assisted reproductive technologies (such as IVF and ICSI). However, these technologies have significant limitations in treating patients with gene defects. Even after obtaining fertilized eggs through artificial insemination, the rate of subsequent embryonic developmental arrest remains high, suggesting that the root cause may lie in the developmental quality of the oocytes themselves, rather than simply obstacles in the fertilization process. Currently, there are no effective interventions in clinical practice to improve oocyte quality.

[0005] Ovarian follicles are the basic structural and functional units of the mammalian ovary, within which oocytes complete their growth, development, and maturation. The normal state of the follicular microenvironment plays a decisive role in the recovery of oocyte meiosis, organelle rearrangement, and the establishment of epigenetic modifications, directly determining the developmental potential of the oocyte and the developmental fate of the embryo after fertilization. Previous studies have shown that damage to oocytes during follicular development caused by adverse factors is often irreversible and continues to affect the preimplantation embryonic stage. However, whether intervention in follicular development can improve the developmental quality of oocytes or embryos associated with NLRP2 gene defects remains unclear.

[0006] Sodium carbenoxolone (CBX) is a semi-synthetic derivative of glycyrrhetinic acid, with the chemical formula C0. 34 H 50O7, also known as gastrin sodium, is mainly used clinically to protect the gastric mucosa and promote ulcer healing. Studies have confirmed that CBX treatment can restore meiosis in oocytes of multiple species, including rats, mice, pigs, and cattle, and alter intracellular cyclic nucleotide levels. However, there are currently no studies exploring whether sodium glycerate can be used to improve infertility caused by NLRP2 gene defects, nor are there any studies investigating whether sodium glycerate improves embryonic development under maternal administration conditions.

[0007] In summary, there is an urgent need in this field to develop a precise intervention program for NLRP2 gene defect-related infertility, especially a safe and effective treatment method that can intervene during oocyte development without direct manipulation of the embryo. Summary of the Invention

[0008] Based on the aforementioned deficiencies in existing technologies, this invention first provides sodium glycinate in the preparation of therapeutic... NLRP2 Application in drugs for infertility caused by genetic defects.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] This invention first provides an improvement NLRP2 The application of drugs to improve oocyte quality and early embryonic development in individuals with NLRP2 gene-deficient infertility, through the administration of sodium carbenoxolone (CBX) to the mother before oocyte maturation, significantly increased blastocyst formation rate and live birth rate. Cross-species validation in cynomolgus monkeys confirmed its therapeutic effect on preimplantation embryo arrest.

[0011] Therefore, this invention first protects sodium glycinate in the preparation of therapeutic... NLRP2 Application in drugs for infertility caused by genetic defects.

[0012] Preferably, in the aforementioned application, the sodium glycinate has the following effects:

[0013] (1) Improve NLRP2 Blastocyst formation rate in individuals with genetic defects, and / or;

[0014] (2) Improve NLRP2 Live birth rate of individuals with genetic defects.

[0015] The present invention described above Nlrp2 Edited mice using an intervention regimen with sodium glycinate; Nlrp2This study provides interventional treatments for pre-maturation maternal oocytes in mice with superovulation, embryo flushing, and live birth outcomes. These interventions aim to improve the oocyte developmental microenvironment, thereby increasing blastocyst formation and live birth rates. It also offers infertility treatment strategies and provides safety assessments for both mother and offspring. Furthermore, it expands the application of sodium glycinate interventions to improve pre-implantation embryonic developmental arrest in some cynomolgus monkeys.

[0016] Preferably, in the application described, the sodium gluconate treatment is performed before the mother's oocytes mature and / or before the mother ovulates and / or during the follicle development stage.

[0017] Preferably, in the aforementioned application, the sodium glycinate is administered during the late estrus phase of the mother's estrus.

[0018] Preferably, in the application described, the sodium gluconate is administered to mice at a dose of 5-10 mg / kg body weight.

[0019] This invention also provides the use of sodium gluconate in the preparation of drugs that improve the blastocyst rate after superovulation in cynomolgus monkeys.

[0020] Preferably, in the application described, the amount of sodium glycinate used is 2.5 mg / kg.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention provides for the first time a sodium glycinate in NLRP2 The application evidence in defective infertility fills a technological gap in this field. It clarifies the effective intervention window as "pre-oocyte maturation / pre-ovulation / follicular development stage": Comparative experiments demonstrate that simply adding sodium glycinate to the embryo culture medium has no therapeutic effect; administration to the mother before oocyte maturation is necessary to significantly improve blastocyst formation rate. Cross-species validation proves the universality and clinical translation potential of the protocol: This invention not only validated the effectiveness of the protocol in mouse models but also verified its ability to significantly improve blastocyst rate in non-human primate (cynomolgus monkey) models. Furthermore, this invention simultaneously validated in vitro developmental indicators (blastocyst formation rate) and in vivo reproductive outcomes (live birth count), forming a complete chain of evidence from embryonic development to final fertility, proving that this protocol not only improves in vitro developmental indicators but also truly enhances fertility.

[0023] Good safety profile: The safety of this invention was verified by detecting maternal liver function indicators (ALT, AST) and monitoring offspring growth and development, providing a safety basis for its clinical translation. Simple operation and strong clinical applicability: The drug delivery method of this invention does not require complex surgical procedures and can be achieved through conventional injection. Compared with traditional IVA technology (which requires laparoscopic surgery to obtain ovarian tissue), it is less invasive, lower in cost, and more operable, making it more suitable for clinical promotion.

[0024] Avoiding ethical risks: The intervention window of this invention is to administer medication to the mother before the oocyte matures, which does not involve direct manipulation of the embryo, thus avoiding the ethical risks associated with the embryo. Attached Figure Description

[0025] Figure 1 for Nlrp2 Comparison of 2-cell, 8-cell, and blastocyst formation in mice after treatment with superovulation ( Figure 1 A) and blastocyst rate statistics ( Figure 1 B); Note: The diagram shows... Nlrp2 Edited mice were injected with sodium glycinate followed by superovulation and mating. Two cells were then cultured in vitro to the blastocyst stage. The results and statistical findings are presented below. Nlrp2 - / - The control group consisted of the injected solvent (physiological saline). Nlrp2 - / - Sodium glycinate was used in the sodium glycinate injection group. Two cells from the same mouse were randomly divided and cultured in an environment of 37℃, 5% CO2, 95% air or 37℃, 5% CO2, 5% O2. Blastocyst rate = number of blastocysts / number of cells in the initial culture × 100%, scale bar = 100 μm.

[0026] Figure 2 for Nlrp2 Editing the blastocyst development of mouse embryos in vitro culture supplemented with sodium glycinate; Note: The figure shows the superovulation treatment of wild-type mice (WT) and... Nlrp2 Statistical results of editing mice, cage co-culture, and in vitro culture of 2 cells to blastocysts, WT: wild-type female mice; Nlrp2 - / - Solvent group: Nlrp2 edited mouse 2 cells were cultured in vitro in embryonic culture medium supplemented with sodium glycinate solvent (physiological saline). Nlrp2 - / - Sodium glycinate culture addition: Nlrp2 Mouse 2 cells were cultured in in vitro embryo culture medium with 10 μmol / L sodium glycinate. Both cells were cultured at 37℃, 5% CO2, and 95% aerosol. The blastocyst rate was calculated as (number of blastocysts / initial number of 2 cells) × 100%.

[0027] Figure 3This is a statistical analysis of litter size in mice after administration of medication at different stages of their estrous cycle; Note: The figures show wild-type mice and... Nlrp2 Results of litter size after various treatments and co-hospitalization of mice, WT: wild-type female mice group; Nlrp2 - / - : Nlrp2 The mouse editor does not handle processing groups; Nlrp2 - / - Injection solvent control: Nlrp2 Edit the mouse group that received an equal volume of solvent (physiological saline) via intraperitoneal injection; Nlrp2 - / - (Sodium glycinate) Groups: Groups treated with intraperitoneal injection of sodium glycinate at different stages of the estrous cycle;

[0028] Figure 4 This image shows the detection of plasma liver function indicators in mice after drug administration; Note: The image shows... Nlrp2 Plasma ALT and AST concentrations in mice were measured 4 months after drug administration. WT: Wild-type female mice; Nlrp2 - / - (Comparison): Nlrp2 The mouse editor does not handle processing groups; Nlrp2 - / - Injection solvent control: Nlrp2 Edit the mouse group that received an equal volume of solvent (physiological saline) via intraperitoneal injection; Nlrp2 - / - (Sodium glycerate injection): Treatment group receiving intraperitoneal injection of sodium glycerate at different stages of the estrous cycle.

[0029] Figure 5 The results of editing live offspring weights in mice at 3-6 weeks of age after drug administration were shown; Note: The figures are for... Nlrp2 Edited rats after drug administration and co-hospitalization, offspring weight measurement results, WT: offspring of wild-type female rats; Nlrp2 - / - (Comparison): Nlrp2 The mouse editor does not process the offspring of the group; Nlrp2 - / - Injection solvent control: Nlrp2 Edit the progeny of the mouse group that received an equal volume of solvent (physiological saline) via intraperitoneal injection; Nlrp2 - / - (Sodium glycinate injection): Offspring of groups treated with intraperitoneal injection of sodium glycinate at different stages of the estrous cycle;

[0030] Figure 6 Comparison of blastocyst rates after drug-induced superovulation in cynomolgus monkeys. Note: The figure shows the comparison of blastocyst rates before and after treatment with sodium glycinate in cynomolgus monkeys with preimplantation embryo arrest. Blastocyst rates for the same monkey are connected by a straight line. Blastocyst rate = number of blastocysts / number of fertilized eggs × 100%. Detailed Implementation

[0031] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0032] Example 1: Sodium glycinate improves Nlrp2 Blastocyst formation rate after superovulation in gene-edited mice

[0033] This example provides an improvement Nlrp2 Edit treatment protocols for blastocyst rate after superovulation in mice.

[0034] In the evening, sexually mature Nlrp2-edited mice were intraperitoneally injected with 8 IU PMSG and 10 mg / kg (mouse body weight) sodium glycinate or an equivalent volume of physiological saline. Forty-eight hours later, 8 IU hCG was injected, and the mice were placed in cages with 12-week-old male mice. The next morning, the treated female mice underwent vaginal plug examination. Mice found with plugs were euthanized and dissected 24 hours later. Two cells were extracted from the oviduct and cultured in vitro in a KSOM until they reached the blastocyst stage. Figure 1 As shown, incubation at 37℃, 5% CO2, and 95% air significantly improved the effect of sodium glycinate treatment. Nlrp2 The developmental capacity of mouse 2 cells was edited. Similarly, culture at 37℃, 5% CO2, and 5% O2 conditions, treatment with sodium glycinate also significantly improved [the developmental capacity]. Nlrp2 Editing mouse 2 cell developmental capacity ( Figure 1 A). On the other hand, it can also be seen that under low oxygen (5% O2) conditions, Nlrp2 The developmental capacity of edited mouse cells was significantly higher than that under normoxic conditions. Figure 1 B).

[0035] To clarify that sodium glycinate's mechanism of action lies in maternal administration rather than direct embryonic action, this experiment compared the effects of maternal administration versus direct embryonic administration. Wild-type mice (WT) and... Nlrp2 Two-cell stage embryos obtained after superovulation in edited mice, with wild-type mouse embryos serving as a culture control. Nlrp2 Two-cell embryos of edited mice were randomly divided into two groups: a solvent control group, in which the same volume of solvent (physiological saline, the same below) as the sodium glycinate treatment group was added to KSOM medium, and cultured at 37°C, 5% CO2, and 95% air; and a sodium glycinate (CBX) treatment group, in which 10 μmol / L CBX was added to KSOM medium, and cultured at 37°C, 5% CO2, and 95% air. The culture results are as follows: Figure 2 As shown, when wild-type mouse embryos are cultured in vitro to the blastocyst stage, Nlrp2The rate of edited mouse blastocysts was significantly lower in both the presence and absence of sodium glycinate treatment compared to wild-type mice, indicating that the addition of sodium glycinate to the culture medium for Nlrp2-edited mouse embryos... Nlrp2 The blastocyst rate of edited mouse embryos did not differ significantly with or without sodium glycinate treatment.

[0036] In summary, the therapeutic effect of sodium glycinate depends on the specific protocol of maternal administration, rather than acting directly on the embryo itself.

[0037] Example 2 Sodium glycinate on Nlrp2 The effect of editing on the number of offspring in mice under natural conditions

[0038] This embodiment combines monitoring the estrous cycle of female mice before medication with internal injection of sodium glycinate to improve the effect of sodium glycinate. Nlrp2 Editing mice to provide treatment strategies for low fertility.

[0039] right Nlrp2 The mice were restrained and their vaginas were irrigated gently to minimize stress or pseudopregnancy in the mothers. The rinsed cells were smeared and fixed on slides. In the evening, the vaginal smears were stained using a Wright-Gymsa staining kit and observed under a microscope to record the estrous cycle stages of the mothers.

[0040] For female mice in the postestrus and interestrus phases, administer an intraperitoneal injection of 5 mg / kg (mouse body weight) of sodium glycinate or an equal volume of physiological saline. After 24 hours, they are placed in the same cage with healthy male mice over 12 weeks of age. After 18 hours of co-occurrence, the male mice are removed and the vaginal plugs are examined.

[0041] For female mice in proestrus and estrus, administer an intraperitoneal injection of 5 mg / kg (mouse body weight) of sodium glycinate or an equal volume of physiological saline. After the injection, place them in a cage with healthy male mice over 12 weeks of age. Remove the male mice 18 hours after the injection and examine the vaginal plugs.

[0042] Female mice with a vaginal plug detected are considered successfully fertilized. After 15 days, observe the abdominal swelling; a noticeably swollen abdomen indicates a successful pregnancy. Change the bedding to clean and allow the mice to give birth without interference. Record the litter size of successfully pregnant mice. Results are as follows: Figure 3 As shown, intraperitoneal injection of sodium glycinate at different stages of the estrous cycle resulted in an increase in the number of offspring. Nlrp2 The number of pups in edited mice was significantly higher in the group receiving the drug during the late estrus stage than in the group not receiving the drug, indicating that the drug is most effective when administered at the early stage of follicular development / before oocyte maturation.

[0043] Example 3: Sodium glycinate rescue Nlrp2 Safety assessment after editing the mouse

[0044] This embodiment evaluates the maternal safety (biochemical-liver function) and offspring safety (pup weight) after drug administration, providing a reference for the safety of sodium gluconate in treating infertility.

[0045] Maternal safety: Blood was collected from the orbital rim of euthanized female mice after a 4-month treatment cycle. Whole blood samples were placed in heparin anticoagulant tubes, gently inverted to mix, and centrifuged at 3000 rpm / min for 15 min at 4°C within 30 minutes. The supernatant was collected and stored at -20°C for ALT and AST detection. Results are as follows: Figure 4 As shown, wild-type mice, untreated. Nlrp2 Edited mice, Nlrp2 edited mice injected with solvent, and mice injected with sodium glycinate Nlrp2 There were no significant differences in ALT and AST levels in the edited mice, suggesting that long-term use of sodium glycinate has no effect on maternal liver function.

[0046] Offspring safety: For wild-type female mice during the drug testing period, Nlrp2 Live offspring of edited female mice (untreated, injected with solvent, or injected with sodium glycinate) were monitored for weight for 4 weeks (from weaning to sexual maturity). Monitoring results are as follows: Figure 5 As shown, at 3 weeks of age, Nlrp2 The issue of overweight offspring from the mother mice improved, and their weight approached that of wild-type offspring. By 6 weeks of age, the weight of the wild-type mice, without treatment, was significantly reduced. Nlrp2 Editing mice, injection solvent group Nlrp2 Edited mice and injected with sodium glycinate Nlrp2 There was no significant difference in the body weight of live offspring of the edited mice. These results indicate that injection of sodium glycinate into the mother improves the pre-pubertal weight distribution in offspring and has no significant effect on subsequent weight development (until sexual maturity).

[0047] Example 4: Effect of sodium glycinate on blastocyst rate after superovulation in cynomolgus monkeys

[0048] This case study investigated the use of sodium glycinate in combination with superovulation treatment to improve the blastocyst rate in cynomolgus monkeys with regular menstrual cycles, good superovulation response (8 or more oocytes retrieved), and low blastocyst development rate after ICSI (less than 20%). Superovulation was initiated by intramuscular injection of rhFSH and 2.5 mg / kg (cynomolgus monkey body weight) sodium glycinate at 8 PM on the first day of menstruation (D1) until 8 AM on the tenth day (D10), with injections twice daily at 8 AM and 8 PM. On the evening of the eleventh day (D11), hCG was injected intramuscularly, and oocytes were retrieved via laparoscopy 34-36 hours later. ICSI was performed on oocytes in metaphase II of meiosis, and fertilized eggs with double pronuclei were cultured in vitro to the blastocyst stage. Blastocyst rate = number of blastocysts / number of fertilized eggs × 100%. Treatment results are as follows. Figure 6 As shown, after combined treatment with sodium glycinate superovulation, the blastocyst rate of cynomolgus monkeys was significantly higher than that of their previous superovulation.

Claims

1. Sodium glycinate in the preparation of therapeutic... NLRP2 Application in drugs for infertility caused by genetic defects.

2. The application according to claim 1, characterized in that, The sodium glycinate has the following functions: (1) Improve NLRP2 Blastocyst formation rate in individuals with genetic defects, and / or; (2) Improve NLRP2 Live birth rate of individuals with genetic defects.

3. The application according to claim 2, characterized in that, The sodium glycinate treatment is administered before the mother's oocytes mature and / or before ovulation and / or during follicular development.

4. The application according to claim 3, characterized in that, The sodium gluconate is administered during the late estrus phase of the mother's estrus.

5. The application according to claim 4, characterized in that, The sodium gluconate was administered to mice at a dose of 5-10 mg / kg body weight.

6. Application of sodium gluconate in the preparation of drugs that improve the blastocyst rate after superovulation in cynomolgus monkeys.

7. The application according to claim 6, characterized in that, The dosage of sodium glycinate is 2.5 mg / kg.

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