WO2023109784A1 - Use of dapagliflozin and analogue thereof in preparation of drug for preventing and treating male reproductive dysfunction - Google Patents

Abstract

The present invention relates to the technical field of biomedicines, and in particular, to a use of an SGLT2 inhibitor in preparation of a drug for preventing and treating male reproductive dysfunction. Research results of the present invention indicate that Dapagliflozin treatment can increase the testis weight and the testis/body weight ratio of db/db mice, and can significantly improve the sperm quality abnormity, the sperm quantity abnormity and the sperm movement abnormity of the db/db mice. According to the present invention, the SGLT2 inhibitor is used for preventing or treating the male reproductive dysfunction for the first time, and a more ideal treatment effect is achieved; and the SGLT2 inhibitor provides a new prevention and treatment option for clinical application to treat male sterility, especially the male sterility caused by diabetes or hyperglycemia, and has extremely high application value and social benefit.

Description

Use of dapagliflozin and its analogs in the preparation of drugs for preventing and treating male reproductive dysfunction

Cross References to Related Applications

This application claims the benefit of Chinese Application No. 202111513681.8 filed on December 13, 2021. Said application number 202111513681.8 is hereby incorporated by reference in its entirety.

technical field

The invention belongs to the technical field of biomedicine. It specifically relates to the use of SGLT2 inhibitors (especially dapagliflozin and its analogues) in the preparation of drugs for preventing and treating male reproductive dysfunction.

Background technique

In recent years, with the aggravation of environmental pollution and the delay of childbearing age, the incidence of male infertility has been increasing year by year. The most direct consequence of the increase in the incidence of infertility is the reduction of the fertility rate, which will further accelerate the process of my country's entry into an aging society. Sustainable development poses serious challenges. The occurrence of infertility is related to many factors, the individual differences are large, and the pathogenic factors are complex, which brings difficulties to the research and treatment of the disease. The incidence of male infertility is related to many factors such as environment, heredity, living habits, and endocrine diseases. Pathological changes mainly include spermatogenesis and maturation disorders, which are generally manifested clinically as oligozoospermia, asthenozoospermia, teratozoospermia and azoospermia. Oligospermia and asthenozoospermia caused by abnormal spermatogenesis and maturation account for more than half of male infertility . The pathogenic mechanism of oligoasthenozoospermia is complex, the population is highly heterogeneous, and there is a lack of effective and targeted treatment measures. Therefore, in-depth exploration of the pathogenic mechanism of spermatogenesis disorders and the development of drugs that can effectively prevent or treat diseases related to spermatogenesis disorders are of great strategic research significance for improving the quality of my country's population and quality of life, and supporting the construction of a "healthy China".

Diabetes is one of the most common clinical metabolic diseases that affect male fertility. In recent years, the onset of diabetes has tended to be younger. Epidemiological surveys show that as of 2014, there are about 420 million people with diabetes worldwide. Therefore, the reproductive age affected by diabetes There are a large number of men in the period, especially the number of children and adolescents with diabetes is increasing year by year. The male reproductive system is one of the more common serious complications of diabetes. The physiological structure and function of the testis in diabetic patients are severely damaged, and the androgen is low, and the sperm count, motility, deformity rate and sperm DNA integrity will be damaged to varying degrees. However, The pathogenic mechanism of diabetes leading to abnormal spermatogenesis has not been fully elucidated. At present, anti-oxidative symptomatic treatment, empirical treatment of traditional Chinese medicine and assisted reproductive technology are mainly used for the decline of male fertility caused by diabetes. The treatment effect of some patients is not good, and there is still a lack of targeted prevention and treatment programs. In view of the large number of diabetic patients worldwide, it is urgent to explore the molecular mechanism of diabetic testicular spermatogenesis dysfunction and male infertility, and to develop drugs that can effectively prevent or treat male reproductive dysfunction caused by diabetes. .

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of hypoglycemic drugs, which can exert hypoglycemic effects by inhibiting the reabsorption of glucose in the proximal tubule of the kidney. As a new target of hypoglycemic drugs, SGLT-2 selective inhibitors have no significant impact on other tissues and organs due to their specific distribution in the kidney; diabetic patients with insulin resistance can still benefit; and they are not prone to hypoglycemia risk, without increasing Diabetic weight and other advantages. At present, there are 6 kinds of SGLT2 inhibitors on the market in the world, namely: Canagliflozin (canagliflozin), Dapagliflozin (dapagliflozin), Empagliflozin (empagliflozin), Ipragliflozin (epagliflozin), Luseogliflozin (luxagliflozin) net) and Tofogliflozin (togegliflozin). In recent years, large-scale cardiovascular outcome studies have shown that a variety of SGLT2 inhibitors, including dapagliflozin, can significantly improve the cardiovascular and renal outcomes of patients with type 2 diabetes [N Engl J Med, 2017, 377 (7 ):644-657.][N Engl J Med,2019,380(24):2295-2306.][N Engl J Med 2019,380(19):1881-1882.]. Therefore, this class of drugs has become the drug of choice for patients with type 2 diabetes mellitus complicated with cardiovascular disease and diabetic nephropathy. However, the effect of these drugs on reproductive function has not been reported yet.

Contents of the invention

In order to solve the serious shortage of clinically effective drugs for the prevention or treatment of male reproductive dysfunction, the inventors discovered for the first time that SGLT2 inhibitors have a therapeutic effect on male infertility animal models, providing a new method for male reproductive dysfunction. prevention and treatment options.

Specifically, the present invention is realized through the technical solutions of the following aspects:

The invention provides the use of the SGLT2 inhibitor in the preparation of medicines for preventing and treating male reproductive dysfunction.

Preferably, the SGLT2 inhibitor is selected from one or more of the following: dapagliflozin, empagliflozin, empagliflozin, rupagliflozin or topagliflozin.

More preferably, the SGLT2 inhibitor is dapagliflozin.

In one embodiment, the male reproductive disorder is a disorder of spermatogenesis.

In a preferred embodiment, said male reproductive dysfunction is abnormal sperm quality, abnormal sperm quantity and/or abnormal sperm motility.

In another preferred embodiment, the male reproductive dysfunction is azoospermia, oligospermia, asthenospermia and/or sperm deformity.

In yet another preferred embodiment, said male reproductive dysfunction is caused by diabetes or hyperglycemia.

Further, the male refers to a male mammal.

Preferably, the mammal is selected from the group consisting of mice, rats, rabbits, cats, dogs or primates.

More preferably, said mammal is selected from humans.

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

In the present invention, SGLT2 inhibitors (especially dapagliflozin and its analogues) are used for the prevention or treatment of male reproductive dysfunction for the first time, and a relatively ideal therapeutic effect is achieved. Male sterility caused by hyperglycemia) provides a new prevention and treatment option, which has high application value and social benefits.

Description of drawings

The accompanying drawings are used to provide a further understanding of the present invention, and constitute a part of the description, and are used together with the embodiments of the present invention to explain the present invention, and do not constitute a limitation to the present invention. In the attached picture:

Figure 1: Changes in body weight and blood glucose levels of mice.

Figure 2: Mouse gross body and testis weight ratio.

Figure 3: HE staining results of mouse testis tissue.

Figure 4: Identification results of germ cell markers in mouse testis tissue.

Figure 5: Detection results of mouse sperm density and survival rate.

Figure 6: Detection results of mouse sperm motility indicators.

Figure 7: Detection results of intrinsic parameters of mouse sperm motility.

Figure 8: TUNEL staining results of mouse testis tissue.

Figure 9: Expression results of apoptosis-related proteins in mouse testis tissue.

Figure 10: Expression results of apoptosis-related proteins in mouse testis tissue.

Figure 11: Results of oxidative stress levels in mouse testis tissue.

Detailed ways

The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, and are not intended to limit the scope of the present invention.

The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field, or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased through regular channels.

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, are commercially available products.

Example:

1. Experimental method:

Classic type 2 diabetic mice: leptin receptor double knockout mice (db/db mice, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), 8-week-old mice were selected, and after 1 week of adaptive feeding, The mice whose random blood glucose was detected to be ≥16.7mmol/L were included in this experiment. The mice were randomly divided into two groups: the diabetes group and the intervention group. Dapagliflozin (Dapa, 1 mg/kg body weight/day) and water (equal volume) were administered to the diabetic group, respectively, orally administered once a day for 5 weeks. Littermate wild-type mice were also selected as normal controls. 9-10 in each group. Determination of fasting body weight, random body weight, fasting blood glucose and random blood glucose of mice before and after intervention

Typical features of leptin receptor-deficient model mice (db/db) include metabolic disturbances such as hyperleptinemia, obesity, hyperglycemia, hyperinsulinemia, decreased prolactin in serum and increased prolactin in pituitary, and typical infertility [Science. 1966 Sep 2; 153(3740): 1127-1128]. Humans with mutations in leptin or its receptors are also obese and infertile [Endocrine reviews. 2006;27:710–718]. The classic animal model of type 2 diabetes, db/db mice, exhibits pathological phenotypes very similar to those of diabetic males in male reproductive system damage, including abnormal spermatogenic function and sexual dysfunction, and is the most commonly used to study male infertility and sexual dysfunction. One of the models [Biochemical and Biophysical Research Communications, 2017; 485:686-692; Diabetologia 2020 09; 63(9)]. Therefore, we chose this typical type 2 diabetic mouse as a model of male testicular tissue injury for research.

Animal experiments strictly followed the animal experiment management regulations of Peking University, and were reviewed by the Animal Experiment Ethics Committee of Peking University.

Sample collection and follow-up experiments were performed on the second day after administration was completed.

(1) Tissue collection: mice were weighed, and testicular tissues were weighed. Keep fresh epididymis. The testicular tissue on one side was fixed with paraformaldehyde, routine alcohol dehydration and paraffin-embedded section; the testicular tissue on the other side was directly extracted for protein or frozen in liquid nitrogen.

(2) Detection of the impact on the morphology of the testicular tissue seminiferous tubules: Paraffin sections of the testicular tissue were subjected to routine histomorphological staining (hematoxylin-eosin staining) to observe the morphological changes of the testicular tissue seminiferous tubules of mice in each group , such as the arrangement of seminiferous tubules, the shape of the lumen, the arrangement and number of spermatogenic cells at all levels, etc. The expression of germ cell marker proteins such as DAZL (spermatogonia), SYCP3 (spermocytes), TNP1 (sperm cells), PGK2 (sperm) and SOX9 (Sertoli cells) were detected by immunofluorescence method.

(3) Determination of the impact on semen quality: fresh epididymis, epididymis sperm collected by diffusion method and computer-aided semen analysis system (Beijing Weili New Century Technology Development Co., Ltd., model: WLJY-9000) to detect the number of sperm and sperm in mice Survival; rapid forward motion and forward motion; linear velocity, curved velocity, mean path velocity, linearity, head roll displacement, and linearity.

(4) Clarify the effect on oxidative stress and apoptosis: Paraffin sections of testis tissue were tested with one-step TUNEL cell apoptosis detection kit (green fluorescence) to detect cell apoptosis in paraffin sections of testis tissue of mice in each group . Fresh or liquid nitrogen-frozen testicular tissue, after protein extraction, Western blot was used to detect the expression of apoptosis markers (including pro-apoptotic proteins and anti-apoptotic proteins), total antioxidant capacity detection kit (ABTS rapid method), Total SOD activity detection kit (NBT method), glutathione peroxidase detection kit (NADPH method), hydrogen peroxide and lipid oxidation (MDA) detection kit were used to detect the total antioxidant capacity; SOD and Glutathione peroxidase activity, hydrogen peroxide and malondialdehyde (Malondialdehyde, MDA) content, Western blot method to detect 4-HNE (4-Hydroxynonenal, 4-hydroxynonenal) protein expression.

(5) All experimental data were analyzed and graphed using GraphPad Prism 7.0 statistical software. Experimental data are expressed as mean ± standard error (mean ± SEM). Differences between multiple groups were compared using analysis of variance and Holm-Sidak post-test, and P<0.05 indicated that the difference was statistically significant. * represents P<0.05; ** represents P<0.01; *** represents P<0.001.

2. Experimental results:

2.1 Changes in body weight and blood sugar levels of mice

First, littermates of wild type mice (wild type/WT mice), leptin receptor double knockout mice (db/db mice) and db/db mice treated with dapagliflozin (Dapa) were tested Fasting body weight and random body weight before treatment and at the end of treatment, it was found (see Figure 1): Compared with WT mice, the body weight of db/db mice and Dapa mice all increased significantly; then each group was detected The blood glucose of the mice was found (see Figure 1): at the beginning of treatment, compared with WT mice, the fasting blood glucose and random blood glucose of db/db mice and Dapa mice were all significantly increased; and at the end of treatment , compared with WT mice, the fasting blood glucose and random blood glucose of db/db mice were significantly increased, and Dapa treatment could significantly reduce the fasting blood glucose and random blood glucose of db/db mice.

2.2 Ratio of gross body weight and testis weight of mice

The body weight, testis weight and testis/body weight ratio of mice in each group were detected at the end of treatment. The results found (see Figure 2): Compared with WT mice, the body weight of db/db mice and Dapa mice all increased significantly; the testis weight and testis/body weight ratio of db/db mice were significantly reduced, and Dapa treatment could Significantly increased testis weight and testis/body weight ratio in db/db mice.

2.3 HE staining results of mouse testis tissue

Hematoxylin-eosin (HE) staining was performed on the testicular tissues of the mice in each group, and it was found (see Figure 3): the spermatogenic cells at all levels in the seminiferous tubules of the WT mice were arranged neatly, and a large number of mature cells could be seen in the lumen. Sperm; in the seminiferous tubules of db/db mice, there are only a small number of germ cells at all levels, and the arrangement is disordered, and mature sperm can hardly be seen in the lumen; in the seminiferous tubules of db/db mice treated with Dapa The spermatogenic cells reappeared and arranged more regularly, and a small amount of spermatozoa could also be seen in the lumen.

2.4 Identification results of germ cell markers in mouse testis tissue

The expression of all levels of germ cell marker proteins in the testis tissues of mice in each group was detected by immunofluorescence. The results showed (see Figure 4): Similar to HE results, compared with WT mice, germ cell markers (DAZL/SYCP3), sperm cell markers (TNP1), and sperm markers in the seminiferous tubules of db/db mice Both markers (PGK2) and Sertoli cell markers (sox9) were significantly decreased; after Dapa treatment, these markers were significantly increased.

2.5 Mouse sperm quality results

The semen quality of the mice in each group was detected by computer-assisted semen analysis, and it was found (see Figure 5): compared with WT mice, the sperm density of db/db mice was significantly lower, and the db/db mice treated with Dapa were less The sperm density of mice was significantly increased; similarly, it was found that the sperm survival rate of db/db mice was significantly reduced, while the sperm density of Dapa-treated mice had a tendency to increase.

At the same time, as shown in Figure 6, the rapid forward movement and forward movement of sperm in db/db mice were significantly reduced, while the rapid forward movement and forward movement of sperm in Dapa-treated mice were significantly increased.

As shown in Figure 7, the intrinsic motility indicators of db/db mouse sperm include linear velocity (VSL), curved velocity (VCL), average path velocity (VAP), linear (LIN) and lateral displacement of the straight sperm head (ALH) were significantly reduced, and Dapa treatment can improve sperm intrinsic motility indicators except for linearity (LIN). Neither db/db nor Dapa treatment has any effect on the linearity of mouse sperm.

2.6 Test results of mouse testis tissue

TUNEL staining was used to detect the apoptosis in the testis tissues of mice in each group, and the results showed (see Figure 8): the apoptosis in the testis tissues of db/db mice increased significantly, while the testis tissues of db/db mice treated with Dapa Cell apoptosis was significantly reduced.

The expression changes of related proteins in two typical pathways affecting cell apoptosis in mouse testis tissue were detected by Western blot. The results found (see Figure 9): the expression of anti-apoptotic protein Bcl2 in the testis tissue of db/db mice was significantly reduced, while the expression of apoptotic protein BAX protein tended to increase; The expression of anti-apoptotic protein Bcl2 tended to increase. The protein expression of Bcl2/BAX was significantly decreased in the testis tissues of db/db mice, while it was significantly upregulated in the testis tissues of Dapa-treated db/db mice.

In another apoptotic pathway, it was found (see Figure 10): the expression of the anti-apoptotic protein XIAP in the testis tissue of db/db mice was significantly reduced, while the expression of the apoptotic protein caspase8 protein tended to increase, and the protein expression of caspase9 protein increased significantly , meanwhile, the expression of anti-apoptotic protein XIAP was significantly up-regulated in the testis tissue of Dapa-treated db/db mice. The protein expression of XIAP/caspase3 tended to decrease in the testis tissues of db/db mice, while it was significantly up-regulated in the testis tissues of Dapa-treated db/db mice; significantly decreased in the testis tissues of Dapa-treated db/db mice; the protein expression of XIAP/caspase9 was significantly decreased in the testis tissues of db/db mice, while in Dapa-treated db/db mice It was significantly up-regulated in testis tissue.

Finally, the changes in the levels of oxidative stress in the testicular tissues of the mice in each group were detected by the method of the kit, and it was found (see Figure 11): the total antioxidant capacity in the testicular tissues of the db/db mice was significantly reduced, and the antioxidant kinase The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) decreased significantly, while the protein expression of oxidative stress product dodecahydroxynonivalenal (4-HNE) increased significantly; at the same time , the total antioxidant capacity in the testis tissue of Dapa-treated db/db mice tended to increase, the activity of the anti-oxidative stress enzyme SOD was significantly up-regulated, the activity of GSH-Px tended to increase, and the protein expression of oxidative stress product 4-HNE is significantly reduced.

3. Experimental conclusion

Dapagliflozin treatment can increase testis weight and testis/body weight ratio in db/db mice, and can significantly improve abnormal sperm quality, abnormal sperm count and abnormal sperm motility in db/db mice.

In summary, the present invention uses SGLT2 inhibitors for the first time in the prevention or treatment of male reproductive dysfunction, and achieves a relatively ideal therapeutic effect. It is clinically used to treat male infertility, especially male infertility caused by diabetes or hyperglycemia , providing a new prevention and treatment option with high application value and social benefits.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims (8)

1. Use of sodium-glucose cotransporter 2 (Sodium-glucose cotransporter 2, SGLT2) inhibitor in the preparation of medicines for preventing and treating male reproductive dysfunction.
2. The use according to claim 1, characterized in that: the SGLT2 inhibitor is selected from one or more of the following: dapagliflozin, empagliflozin, empagliflozin, rupagliflozin or toglie clean up.
3. The use according to claim 1 or claim 2, characterized in that: the SGLT2 inhibitor is dapagliflozin.
4. The use according to claim 1 or claim 2, characterized in that: the male reproductive dysfunction is spermatogenesis disorder.
5. The use according to claim 1 or claim 2, characterized in that: the male reproductive dysfunction is abnormal sperm quality, abnormal sperm quantity and/or abnormal sperm motility.
6. The use according to claim 1 or claim 2, characterized in that: the male reproductive dysfunction is azoospermia, oligospermia, asthenospermia and/or sperm deformity.
7. The use according to claim 1 or claim 2, characterized in that: the male reproductive dysfunction is caused by diabetes or hyperglycemia.
8. The use according to claim 1 or claim 2, characterized in that: the male refers to a male mammal, preferably, the mammal is selected from the following: mice, rats, rabbits, cats, dogs or spirits In the long category, more preferably, said mammal is selected from humans.

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